U.S. patent application number 16/620627 was filed with the patent office on 2020-06-18 for combination therapy.
The applicant listed for this patent is GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED. Invention is credited to Meixia BI, Christopher B. HOPSON, Patrick A. MAYES, Sapna YADA VILLI.
Application Number | 20200190194 16/620627 |
Document ID | / |
Family ID | 71073396 |
Filed Date | 2020-06-18 |
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United States Patent
Application |
20200190194 |
Kind Code |
A1 |
BI; Meixia ; et al. |
June 18, 2020 |
COMBINATION THERAPY
Abstract
The present invention provides methods of treating cancer in a
patient in need thereof, the method comprising administering to the
patient an effective amount of an agent directed to human ICOS and
an effective amount of an agent directed to human PD1 or human
PD-L1 sequentially. The present invention also provides an
anti-ICOS antibody or antigen binding fragment thereof and an
anti-PD1 antibody or antigen binding fragment thereof for
sequential use in treating cancer in a human in need thereof. The
present invention provides an anti-ICOS antibody or antigen binding
fragment thereof and an anti-PD-L1 antibody or antigen binding
fragment thereof for sequential use in treating cancer in a human
in need thereof.
Inventors: |
BI; Meixia; (Collegeville,
PA) ; HOPSON; Christopher B.; (Collegeville, PA)
; MAYES; Patrick A.; (Devon, PA) ; YADA VILLI;
Sapna; (Collegeville, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE INTELLECTUAL PROPERTY DEVELOPMENT LIMITED |
BRENTFORD |
|
GB |
|
|
Family ID: |
71073396 |
Appl. No.: |
16/620627 |
Filed: |
June 8, 2018 |
PCT Filed: |
June 8, 2018 |
PCT NO: |
PCT/IB2018/054167 |
371 Date: |
December 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62517309 |
Jun 9, 2017 |
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62662278 |
Apr 25, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2818 20130101;
A61P 35/00 20180101; C07K 16/2827 20130101; A61K 2039/505 20130101;
C07K 2317/76 20130101; C07K 2317/52 20130101; C07K 2317/75
20130101; C07K 2317/73 20130101; A61K 2039/507 20130101; C07K
2317/24 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating cancer in a patient in need thereof, the
method comprising administering to the patient an effective amount
of an agent directed to human ICOS and an effective amount of an
agent directed to human PD1 or human PD-L1 sequentially, wherein
administration of the agent directed to human ICOS is followed by
administration of the agent directed to human PD1 or human
PD-L1.
2. The method of claim 1, wherein the agent directed to human ICOS
is an anti-ICOS antibody or antigen binding portion thereof.
3. The method of claim 2, wherein the anti-ICOS antibody is an ICOS
agonist.
4. The method of claim 2, wherein the anti-ICOS antibody comprises
a V.sub.H domain comprising an amino acid sequence at least 90%
identical to the amino acid sequence set forth in SEQ ID NO:7; and
a V.sub.L domain comprising an amino acid sequence at least 90%
identical to the amino acid sequence as set forth in SEQ ID
NO:8.
5. The method of claim 2, wherein the anti-ICOS antibody comprises
a V.sub.H domain comprising the amino acid sequence set forth in
SEQ ID NO:7 and a V.sub.L domain comprising the amino acid sequence
as set forth in SEQ ID NO:8.
6. The method of claim 1, wherein the agent directed to human PD1
is an anti-PD1 antibody or antigen binding portion thereof.
7. The method of claim 6, wherein the anti-PD1 antibody is a PD1
antagonist.
8. The method of claim 6, wherein the anti-PD1 antibody is
pembrolizumab.
9. The method of claim 6, wherein the anti-PD1 antibody is
nivolumab.
10. The method of claim 1, wherein the agent directed to human
PD-L1 is an anti-PD-L1 antibody or antigen binding portion
thereof.
11. The method of claim 10, wherein the anti-PD-L1 antibody is a
PD1 antagonist.
12. The method of claim 1, wherein the agent directed to human ICOS
or anti-ICOS antibody or antigen binding portion thereof is
administered once every week, once every two weeks, once every
three weeks, or once every four weeks.
13. The method of claim 1, wherein the agent directed to human PD1
or human PD-L1 or anti-PD1 antibody or antigen binding portion
thereof or anti-PD-L1 antibody or antigen binding portion thereof
is administered once every week, once every two weeks, once every
three weeks, or once every four weeks.
14. The method of claim 1, wherein the cancer is selected from the
group consisting of colorectal cancer (CRC), gastric, esophageal,
cervical, bladder, breast, head and neck, ovarian, melanoma, renal
cell carcinoma (RCC), EC squamous cell, non-small cell lung
carcinoma, mesothelioma, pancreatic, and prostate cancer.
15. The method of claim 1, wherein the agent directed to human
ICOS, or anti-ICOS antibody or antigen binding portion thereof, is
administered as an intravenous (IV) infusion.
16. The method of claim 1, wherein the agent directed to human PD1
or human PDL1, or anti-PD1 antibody or antigen binding portion
thereof or anti-PDL1 antibody or antigen binding portion thereof,
is administered as an intravenous (IV) infusion.
17. The method of claim 1, wherein the start of administration of
the agent directed to human PD1 or human PDL1, or anti-PD1 antibody
or antigen binding portion thereof or anti-PDL1 antibody or antigen
binding portion thereof, is initiated at a time point selected from
1 week, 2 weeks, 3 weeks, and 4 weeks after the start of the
administration of the agent directed to human ICOS, or anti-ICOS
antibody or antigen binding portion thereof.
18. The method of claim 1, wherein the agent directed to human
ICOS, or the anti-ICOS antibody or antigen binding portion thereof,
and the agent directed to human PD1 or human PDL1, or the anti-PD1
antibody or antigen binding portion thereof or the anti-PDL1
antibody or antigen binding portion thereof, are administered to
said human until said human shows disease progression or
unacceptable toxicity.
19.-40. (canceled)
41. A method of treating cancer comprising administering an
anti-ICOS antibody or antigen binding portion thereof and an
anti-PD1 antibody or antigen binding portion thereof, wherein the
anti-ICOS antibody or antigen binding portion thereof and an
anti-PD1 antibody or antigen binding portion thereof are
sequentially administered, and wherein administration of the
anti-ICOS antibody or antigen binding portion thereof is followed
by administration of the anti-PD1 antibody or antigen binding
portion thereof.
42. A method of treating cancer comprising administering an
anti-ICOS antibody or antigen binding portion thereof and an
anti-PDL1 antibody or antigen binding portion thereof, wherein the
anti-ICOS antibody or antigen binding portion thereof and an
anti-PDL1 antibody or antigen binding portion thereof are
sequentially administered, and wherein administration of the
anti-ICOS antibody or antigen binding portion thereof is followed
by administration of the anti-PDL1 antibody or antigen binding
portion thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to immunotherapy in
the treatment of human disease. More specifically, the present
invention relates to the use of sequenced dosing of
immunomodulators such as anti-ICOS antibodies, anti-PD1 antibodies,
and anti-PDL1 antibodies in the treatment of cancer.
BACKGROUND OF THE INVENTION
[0002] Cancer immunity is a multistep process that is tightly
regulated by a series of negative immune checkpoint and positive
co-stimulatory receptors that when effectively triggered can
achieve antitumor response (Mellman, I., et al. (2011) Cancer
Immunotherapy Comes of Age. Nature 480(7378), 480-489). However,
tumors have established various mechanisms to circumvent immune
clearance by altering the responsiveness of the immune infiltrate.
In some instances, tumors will be highly dependent on a single
mechanism, and in these cases, there is the potential to achieve
significant clinical activity with single agent immunomodulatory
therapy (Hoos, A. (2016). Development of immuno-oncology
drugs--from CTLA4 to PD1 to the next generations. Nat Rev Drug
Discov. 15(4), 235-47). However, as tumors often utilize multiple,
overlapping and redundant mechanisms to block antitumor immune
response, combination therapy will likely be required for durable
efficacy across a wide range of tumor types. Therefore, new immune
targeted therapies are needed to improve the treatment of all
cancers.
[0003] Thus, there is a need for combination treatments and
strategies for dosing of immunomodulators for the treatment of
disease, in particular cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 is a table showing the study design of anti-ICOS
antibody/anti-PD1 antibody concurrent and phased dosing study
described herein.
[0005] FIG. 2 is a schematic showing the study procedure of
anti-ICOS antibody/anti-PD1 antibody concurrent and phased dosing
study described herein. Shown at the bottom of FIG. 2 is a table
listing antibodies used in the study.
[0006] FIG. 3 is a plot showing average tumor volume of mice groups
treated with anti-ICOS antibody and anti-PD1 antibody concurrently
or in sequential phases (e.g., lead-in dose/follow-up dose) and
group(s) treated with control(s), as indicated in the figure
legend.
[0007] FIG. 4 is a plot showing average tumor volume of mice groups
treated with concurrent dosing of anti-ICOS antibody and anti-PD1
antibody, and group(s) treated with control(s), as indicated in the
figure legend.
[0008] FIG. 5 is a plot showing average tumor volume of mice groups
treated with phased dosing of anti-ICOS antibody and anti-PD1
antibody, and group(s) treated with control(s), as indicated in the
figure legend.
[0009] FIG. 6 is a plot showing average tumor volume of mice groups
treated with phased dosing of anti-PD1 antibody and anti-ICOS
antibody, with anti-PD1 antibody as lead-in dose and anti-ICOS
antibody as follow-up dose, and group(s) treated with control(s),
as indicated in the figure legend.
[0010] FIG. 7 is a plot showing average tumor volume of mice groups
treated with phased dosing of anti-ICOS antibody and anti-PD1
antibody, with anti-ICOS antibody as lead-in dose and anti-PD 1
antibody as follow-up dose, and group(s) treated with control(s),
as indicated in the figure legend.
[0011] FIGS. 8A-8C are sets of plots showing individual tumor
volumes of mice treated with concurrent dosing of anti-ICOS
antibody and anti-PD1 antibody, and group(s) treated with
control(s), as indicated in the corresponding figure legend(s).
FIG. 8A shows individual tumor volumes of mice in Group 1 (left)
and Group 2 (right). FIG. 8B shows individual tumor volumes of mice
in Group 3 (top left), Group 4 (top right), and Group 5 (bottom).
FIG. 8C shows individual tumor volumes of mice in Group 6 (left)
and Group 7 (right).
[0012] FIGS. 9A-9C are sets of plots showing individual tumor
volumes of mice treated with phased dosing of anti-ICOS antibody
and anti-PD1 antibody, and group(s) treated with control(s), as
indicated in the corresponding figure legend(s). FIG. 9A shows
individual tumor volumes of mice in Group 1 (left) and Group 2
(right). FIG. 9B shows individual tumor volumes of mice in Group 8
(top left), Group 9 (top right), and Group 10 (bottom).
[0013] FIG. 9C shows individual tumor volumes of mice in Group 11
(top left), Group 12 (top right), and Group 13 (bottom).
[0014] FIG. 10 is a plot showing survival of mice in all groups
(Groups 1-13). Mice in the groups were treated with concurrent or
phased dosing of anti-ICOS antibody and anti-PD1 antibody or
treated with control(s), as indicated in the figure legend.
[0015] FIG. 11 is a plot showing survival of mice groups treated
with concurrent dosing of anti-ICOS antibody and anti-PD1 antibody,
and group(s) treated with control(s), as indicated in the figure
legend.
[0016] FIG. 12 is a plot showing survival of mice groups treated
with phased dosing of anti-ICOS antibody and anti-PD1 antibody, and
group(s) treated with control(s), as indicated in the figure
legend.
[0017] FIG. 13 is a plot showing survival of mice groups treated
with phased dosing of anti-PD1 antibody and anti-ICOS antibody,
with anti-PD1 antibody as lead-in dose and anti-ICOS antibody as
follow-up dose, and group(s) treated with control(s), as indicated
in the figure legend.
[0018] FIG. 14 is a plot showing survival of mice groups treated
with phased dosing of anti-ICOS antibody and anti-PD1 antibody,
with anti-ICOS antibody as lead-in dose and anti-PD1 antibody as
follow-up dose, and group(s) treated with control(s), as indicated
in the figure legend.
[0019] FIG. 15: Development of an anti-human ICOS agonist
monoclonal antibody
(A) H2L5 binding to dimeric human ICOS (B) human ICOS-L binding to
dimeric human ICOS (C) Binding of H2L5 (20 .mu.g/mL) to CD4
(**P=0.0011, t=4.183, df=13) and CD8 (**P=0.0078, t=3.686, df=7) T
cells from healthy donors. Each symbol represents a separate human
donor, horizontal lines indicate median, and bars are interquartile
range (D) Representative Western Blot demonstrating induction of
AKT signaling in purified activated T cells after treatment with
H2L5 (E) Quantification of CD69+CD4 (*P=0.0142, t=3.416 df=6) or
CD8 (**P=0.0012, t=5.734 df=6) T cells and (F) quantification of
Ki67.sup.+ CD4 (*P=0.0190, t=3.809 df=4) or CD8 (*P=0.0255, t=3.474
df=4) T cells from healthy donor PBMC treated with (12.5 .mu.g/mL)
of bound H2L5 and anti-CD3 for 48 hours. (G, H) Quantification of
soluble IFN-.gamma. from (G) the culture supernatant of PBMC from
healthy subjects treated with (12.5 .mu.g/mL) of bound H2L5 and
anti-CD3 for 24 hours **P=0.0041, t=4.510 df=6 or 48 hrs *P=0.0375,
t=2.661 df=6 (H) the supernatant of NSCLC cancer patient PBMC
treated with (10 .mu.g/mL) bound H2L5 and anti-CD3 for 72 hours.
(I, K) Quantification of RNA expression of (I) T-Bet (TBX21)
(*P=0.0156, t=2.974 df=9) and (J) Granzyme B (GZMB) (**P=0.0020,
t=4.292 df=9) (K) L-Selectin (SELL) (*P=0.0161, t=2.955 df=9) from
healthy donor CD3.sup.+ T cells following indicated treatments
analyzed by a two-tailed, unpaired t-test. H2L5 induces
concentration dependent increases in cytokine production and T-cell
activation from disaggregated tumor cell suspensions. Plates were
coated with H2L5+/-anti-CD3 or isotype control. (L) IFN-.gamma.,
(M) CD8+ OX40+, (N) CD8+ CD25+ following 6 days of culture.
Bars=Group Medians p<0.05 by One Way Anova, **P<0.05
***P<0.0005, ****P<0.000 by One Way Anova. Dashed
line=CD3+isotype IgG4 10 .mu.g/mL. See FIG. S7 for tumor types.
[0020] FIG. 16: Antibody isotype and Fc.gamma.R-engagement is
critical for H2L5 function
(A) PBMC from healthy subjects treated with soluble H2L5 of varying
isotypes at 5 .mu.g/ml for 6 days. Proliferation as measured by
CFSE dilution relative to isotype control (fold change) (B, C)
PBMCs from healthy subjects, with or without depletion of NK cells;
treated with (B) soluble H2L5 of varying isotypes at (5 .mu.g/mL)
for 6 days. (C) Soluble H2L5 of varying isotypes (10 .mu.g/mL) for
24 hours and percentage of dead cells determined by flow cytometry
using NIR Live/Dead dye. An anti-CD52 antibody known to induce
ADCC-mediated T-cell killing was included as a positive control.
(D) ICOS Expression on freshly dissociated patient TIL. The median
fluorescent intensity of ICOS from CD4, CD8, T.sub.reg, and
T.sub.eff cell populations. (Tumor types Solid Triangle=NSCLC (6)
Solid Circle=CRC (4) Solid Diamond=Bladder (2) Solid
Square=Head/Neck (1) Open Triangle=RCC (4) Open Circle=Endometrial
(2) Open Diamond=Prostate (1) Open Square=Thyroid (1); p<0.05 by
One Way Anova). Insert shows histogram of ICOS expression on CD4
(Red), CD8 (orange) and T.sub.reg (Blue) from a patient with
endometrial cancer. (E) Spearman correlation between total ICOS
receptor numbers (calculated by multiplying the percent ICOS
positive for each cell type by the ICOS receptor number per
positive cell) and Fc.gamma.RIIIA reporter assay fold induction in
target cells isolated from PBMCs and patient tumors in presence of
H2L5 IgG1 isotype relative to isotype control for all samples where
both data points were available (r.sup.2=0.681, p<0.001). (F)
Fold induction observed in an Fc.gamma.RIIIA reporter assay using
target cells isolated from NSCLC patient tumor 5001003 incubated
with anti-ICOS antibodies. CD4 T.sub.eff, CD8 T cells and T.sub.reg
were isolated from a dissociated patient tumor and utilized as
target cells in the Fc.gamma.RIIIA assay.
[0021] FIG. 17: H2L5 exhibits FcR dependent agonism to induce
T-cell activation
(A) Isolated CD4 T cells from healthy subjects treated with
indicated concentrations of H2L5 for 60 hours (bound isotype vs.
bound H2L5 ***P=0.0006, t=9.777 df=4, soluble isotype vs. soluble
H2L5 ***P=0.0003, t=11.50 df=4 and (#) bound H2L5 vs. soluble H2L5
**P=0.0017, t=7.530 df=4) (B) PBMC from a healthy subject treated
with soluble H2L5 (ICOS IgG4PE) or H2L5 Fc-disabled at (10
.mu.g/mL) for 3.5 days (isotype control vs. H2L5 **P=0.0056,
t=5.426 df=4), (H2L5 vs. H2L5 Fc-disabled **P=0.0012, t=8.297 df=4)
(C) MLR with anti-CD3 antibody followed by treatment with soluble
H2L5 or H2L5 Fc-disabled antibody at (10 .mu.g/mL) (isotype control
vs. H2L5 *P=0.0166, t=3.966 df=4), (H2L5 vs. H2L5 Fc-disabled
*P=0.0158, t=4.022 df=4) (D) Isolated T cells cultured with and
without monocytes from the same donor followed by treatment with
soluble H2L5 or H2L5 Fc-disabled at (10 .mu.g/mL)+/-anti-CD32 or
Fc-blocking antibody for 4 days. (#) ***P=0.0009, t=8.734 df=4, ($)
**P=0.0031, t=6.405 df=4, (&) *P=0.0389, t=3.026 df=4, (@)
isotype control vs. H2L5 **P=0.0027, t=6.612 df=4, H2L5 vs. H2L5
Fc-disabled *P=0.0239, t=3.544 df=4, H2L5 (control) vs. H2L5
(anti-CD32) **P=0.0066, t=5.184 df=4, H2L5 (anti-CD32) vs. H2L5 (Fc
block) **P=0.0013, t=8.047 df=4 and H2L5 (control) vs. H2L5 (Fc
block) *P=0.0446, t=2.889 df=4. (E, F) Human T cells pre-stimulated
with anti-CD3 for 48 hours and added to a co-culture with human DC.
AlexaFlour488-labeled H2L5 IgG4PE added at 3 .mu.g/mL to
co-cultures on ice then moved to 37.degree. C. for indicated
timepoints. Arrows indicate T cells activated in response to H2L5
treatment, polarization and mobilization towards neighboring
dendritic cell. Data representative of three separate experiments
performed using different donor cells.
[0022] FIG. 18: H2L5 induces an EM phenotype and anti-tumor
activity in humanized mouse model.
(A) Quantification of human CD45.sup.+CD3.sup.+ cells in the blood
of mice H2L5 treatments as compared to isotype control IgG4PE
(****P=<0.0001, F=33.57, df=24) (B) Quantification of human
CD45.sup.+CD3.sup.+CD69.sup.+ cells from the blood of mice H2L5
(1.2 mg/kg) vs. isotype control IgG4PE (*P=0.0119, F=4.179, df=24)
(C) Percentage of CD4 T.sub.CM (0.04 mg/kg **P=0.0038, 0.4 mg/kg
***P=0.0002, 1.2 mg/kg ***P=0.0005, F=8.172, df=20. This is
equivalent to 0.8, 8 and 24 .mu.g per mouse respectively. (D)
CD8.sup.+ T.sub.naive/terminally differentiated effector memory
T.sub.TEMRA (0.004 mg/kg **P=0.0036, 0.04 mg/kg and 0.4 mg/kg
****P=<0.0001, 1.2 mg/kg **P=0.0072, F=13.78, df=20) in the
spleen of mice (E) The percentage of ICOS+ or PD-1+ T cells in mice
implanted subcutaneously with A549 tumor and identified by using PE
conjugated mouse anti human IgG4 by flow cytometry. (F) The ratio
of CD8/T.sub.reg cells in whole tumor tissues (G) HCT116 tumor
volumes on day 13 (0.04 mg/kg) *P=0.0273, (0.4 mg/kg) *P=0.0432,
F=2.788, df=36 (H) A549 tumor volumes on day 21 (0.4 mg/kg)
*P=0.0056, F=3.906, df=36 (i) Kaplan-Meier survival curve of human
PBMC engrafted NSG mice with A549 tumors (A-I) horizontal lines
represent median values, error bars represent interquartile range.
All statistical tests were one-way ANOVA.
[0023] FIG. 19: The isotype of the murine ICOS mAb influences
efficacy in syngeneic tumors.
(A) Kaplan-Meier plots of mice with murine (A) EMT6 murine (B) CT26
syngeneic tumors treated with indicated doses (5, 100 or 200 .mu.g
corresponding to 0.5, 5 and 10 mg/Kg respectively of murine IgG1 or
IgG2a versions of 7E.17G9 antibody twice weekly for 3 weeks or
isotype control (200 .mu.g or 10 mg/kg). Results are representative
of two repeat experiments. Each symbol represents an individual
mouse. Horizontal lines represent median values, error bars
represent interquartile range. All statistical tests were one-way
ANOVA, followed by specific treatment comparators. (C) The ratio of
CD8.sup.+/T.sub.reg in EMT6 or CT26 tumors determined at tumor size
100 mm.sup.3; (D) The percentage of ICOS+ CD4, CD8 and T.sub.reg
cells in tumors (closed circles) or spleens (open circles) of mice
implanted with EMT6 tumors; MFI of ICOS on CD8, CD4 and T.sub.reg
in tumors (closed circles) or spleen (open circles) in mice
implanted with (E) CT26 or (F) EMT6 tumors at tumor sizes of 100
mm.sup.3. (G) Histogram of representative flow plot comparing MFI
of ICOS expression on CD4, CD8 and T.sub.reg isolated from EMT6 and
CT26 tumors; (H) Absolute number of TCR clones expanded in
post-treatment with anti-ICOS 7E.17G9 blood that were also found in
EMT6 tumor (10 .quadrature.g *P=0.0173 and 100 g *P=0.0483; F=3.269
df=28).
[0024] FIG. 20: Evaluation of ICOS expression on different cell
types in human cancers. (A) Expression of ICOS, ICOS-L and PD-L1 in
different tumor types ranked by expression of ICOS from TCGA
database. (B) The expression of ICOS+ cells by single plex IHC and
correlation with expression of PD-L1, PD-1, CD4, CD8, FOXP3 and CD3
in NSCLC. (C) % CD45+ cells that are CD3+, B cells, monocytes, NK
cells, macrophages, dendritic cells in disaggregated tumors from
different solid tumor types. Solid Triangle=NSCLC (6) Solid
Circle=CRC (4) Solid Diamond=Bladder (2) Solid Square=Head/Neck (1)
Open Triangle=RCC (4) Open Circle=Endometrial (2) Open
Diamond=Prostate (1) Open Square=Thyroid (1). (D) The percentage
CD3+CD8+, CD3+CD4+Foxp3+ (T.sub.reg) and the ratio of CD3+, CD8+:
CD3+CD4+Foxp3 in different tumor types. Horizontal line shows
median. (E) Quantification of the co-expression of CD3+PD-1+ICOS+
cells in tumor biopsies obtained from different tumour types by
multiplex IHC. (F) Multiplex IHC of a Head and neck FFPE tumor
sample co-stained for CD3, PD-1 and ICOS (G) Heatmap summarizing
the differentially expressed genes in purified human T cells
treated with H2L5 plus anti-CD3 mAb compared to anti-CD3 alone as
determined by NanoString nCounter analysis System using Human
PanCancer-Immune profiling panel (N=6 donors). (H) Gene expression
changes (fold increase) common between anti-CD3 (0.6 .mu.g/mL) plus
H2L5 (10 .mu.g/mL) activated human T cells (n=6 donors) and murine
EMT6 transplantable tumors after surrogate anti-ICOS (7E.17G9 rat
IgG2b) treatment.
[0025] FIG. 21: ICOS agonist mAb induce PD-1/PD-L1 expression and
enhances activity of anti-PD-1
(A) Quantification of RNA expression of PD-L1 (CD274) (10 .mu.g
*P=0.0137 and 100 .mu.g *P=0.0374; F=5.175 df=10) and (B) PD-1
(Pdcd1) (10 .mu.g *P=0.0194 and 100 .mu.g P=0.1626; F=3.911 df=10)
in EMT6 following indicated treatments. Each symbol represents an
individual mouse sample, horizontal lines represent median values,
error bars represent interquartile range. All statistical tests
were one-way ANOVA with square root transformed data to stabilize
variances (C) Percentage of CD4.sup.+PD-1.sup.+ and
CD8.sup.+PD-1.sup.+ T cells following treatment with isotype
control or H2L5 at 10 .mu.g/mL for 72 hours in PBMC from cancer
patients CD4.sup.+ *P=0.0128, t=3.026 df=10; CD8.sup.+ **P=0.005,
t=3.548, df=10. two-tailed, unpaired t-tests (D). Percentage of
CD4+ ICOS+ in NSCLC or melanoma patients pre- and post-PD-1 therapy
(either pembrolizumab or nivolumab) compared with healthy subjects.
(E) Mice with EMT6 tumors treated with 7E.17G9 IgG1 (10 .mu.g
equivalent to 0.5 mg/kg), anti-PD-1 (200 .mu.g equivalent to 10
mg/kg) or the combination of 7E.17G9 and anti-PD-1 dosed
concomitantly, twice weekly for 3 weeks. (N=10 per treatment group)
(F) A549 tumor volume in NSG mice reconstituted with human PBMC and
treated with H2L5 at 0.8 .mu.g mouse equivalent to 0.04 mg/kg,
isotype 0.8 .mu.g equivalent to 0.04 mg/kg or anti-PD-1
(pembrolizumab/Keytruda) 100 .mu.g equivalent to 5 mg/kg or the
combination of both antibodies. (G) Quantification of IFN-.gamma.
from disseminated NSCLC patient tumors treated with anti-CD3 and
H2L5 (10 .mu.g/mL) for 24 hours. (#) **P=0.0100 ($)
****P=<0.0001 (&) ***P=0.002, F=15.8, df=20. Horizontal
lines represent median values, error bars represent interquartile
range (H) MLR assay evaluating ICOS+pembrolizumab vs. ICOS
**P=0.0036, IgG4PE ICOS+pembrolizumab vs. pembrolizumab **P=0.0090,
ICOS+pembrolizumab vs. 2.times. IgG4PE ***P=0.0009, F=7.324, df=10.
Bars represent mean of triplicate measurement and error bars
represent standard deviation (C-E) All statistical tests were
one-way ANOVA)
[0026] FIG. 22: H2L5 IgG4PE epitope binding (A) An ICOS-L
competition assay by MSD demonstrates that H2L5 IgG4PE partially
competes with ICOS-L for binding to human ICOS receptor. (B)
Activated T cells were incubated with different concentrations of
recombinant ICOS-L (R&D systems) and then incubated with H2L5
and MFI of ICOS CD4+ and CD8+ cells determined by flow
cytometry.
[0027] FIG. 23: H2L5IgG4PE causes dose dependent increases in (A)
cytokine production IFN.gamma., IL-17, IL-10, IL-4, IL-13, IL-5,
IL-2, IL-6, TNF.alpha. measured by MSD (B) activation marker OX40,
CD25 and CD69 on CD4 and CD8 T cells. PBMC were cultured for 48 h
with anti-CD3 (0.6 ug/ml) and different concentrations of
H2L5IgG4PE or isotype control and supernatants harvested for
cytokine analysis and cells for flow cytometry.
[0028] FIG. 24: H2L5 induces concentration dependent increases in
cytokine production from disaggregated tumor cell suspensions from
different cancer patients. Disaggregated tumor cells suspensions
were cultured with plate bound H2L5IgG4PE or isotype control in the
presence or absence of anti-CD3 following 6 day in vitro
stimulation with plate bound anti-CD3 (0.6 .mu.g/mL) and IL2 (100
ng/mL) followed by analysis of (A) IL17, (B) IL10, (C) IL5, (D)
IL13 cytokines in the supernatants by MSD.
[0029] FIG. 25: H2L5 induces concentration dependent increases on
percentage of (A) CD8+LAG3+, p<0.005 by One Way Anova (B) CD8+
PD-1+, (C) ICOS L+cells and (D) (CD4+, CD25+ Foxp3+) p<0.05 by
One Way Anova from disaggregated tumor cell suspensions from
different cancer patients. Disaggregated tumor cells suspensions
were cultured with plate bound H2L5 (ICOS) IgG4PE or isotype
control in the presence or absence of anti-CD3 following 6 day in
vitro stimulation with plate bound anti-CD3 (0.6 .mu.g/mL) and
IL-2(100 ng/mL) followed by flow cytometry. Dashed line=CD3+IgG4
isotype 10 .mu.g/mL Horizontal bars represent median.
[0030] FIG. 26: H2L5 IgG1 induces signaling via the major
activating Fc.gamma.R (Fc.gamma.RIIIa) responsible for ADCC in
humans. (A) Treatment of Jurkat-Fc.gamma.RIIIA-NFAT-luciferase
effector cells and primary human CD4.sup.+ T cells at a ratio of
6:1 with soluble H2L5 of varying isotypes for 6 hrs. An anti-CD52
antibody known to induce ADCC-mediated T cell killing was included
as a positive control (B) Treatment of
Jurkat-Fc.gamma.RIIIA-NFAT-luciferase effector cells and purified
primary human ex vivo tumor derived CD4, CD8 and Tregs at a ratio
of 6:1 with soluble H2L5 IgG1 for 6 hrs Fold change in luciferase
induction produced by Jurkat-Fc.gamma.RIIIA-NFAT-luciferase
effector cells relative to isotype control.
[0031] FIG. 27: H2L5 causes dose dependent binding to ICOS
expressing T cells in blood and tumor. The percentage of ICOS+ or
PD-1+ T cells in whole blood (A) and tumor tissues (B) within each
group, 48 hours post 4.sup.th dose identified using PE conjugated
mouse anti human IgG4 by flow cytometry. Bars represent the median
values for each group.
[0032] FIG. 28: Characterization of an anti-murine ICOS agonist
antibody. Anti-mouse ICOS agonist antibody (7E.17G9) induces
IFN.gamma. production in disseminated mouse splenocytes cultured ex
vivo for 60 hours.
[0033] FIG. 29: Tumor growth for (A) EMT6 or (B) CT26 murine
syngeneic tumors treated with 10 (0.5 mg/kg), 100 (5 mg/kg) or 200
.mu.g (10 mg/kg) doses of murine IgG1 or Ig2a variants of 7E.17G9
antibody or isotype control (200 g (10 mg/kg) twice weekly for 3
weeks. *(numbers) indicate the number of mice with minimally
detectable or non-detectable tumors at study endpoint.
[0034] FIG. 30: % ICOS+ cells within CD4, CD8 and T.sub.reg
populations in tumors (closed circles) and spleens (open circles)
of mice bearing .about.100 mm3 CT26 tumors.
[0035] FIG. 31: (A) absolute number of TCR clones contracted in
post-treatment with Anti-ICOS 7E17G9 antibody blood relative to
pre-treatment blood (10 .mu.g *P=0.0327 and 100 .mu.g *P=0.0497;
F=3.033 df=28) (B) absolute number of TCR clones expanded in
post-treatment blood relative to pre-treatment blood (10 .mu.g
P=0.0975 and 100 .mu.g P=0.1915; F=1.958 df=28) (C) Mean T cell
fraction estimate vs Mean productive clonality
[0036] FIG. 32: Expression of ICOS positive cells in NSCLC, Breast
cancer and CRC by IHC singleplex Immunohistochemical detection of
ICOS in non-small cell lung cancer (NSCLC), breast cancer (BrCA)
TNBrCa, and colorectal cancer (CRC), using a rabbit anti-human
CD278 Monoclonal antibody clone SP98 (Spring Biosciences). Assay
was carried out on the Leica Bond RX with associated platform
reagents. DAB (3, 3'-diaminobenzidine) was used for target
detection. Sections were counter stained with Hematoxylin (All
scale bars=20 um).
[0037] FIG. 33: Changes on cytokine levels from healthy human donor
PBMC in response to treatment with anti-CD3 plus isotype control or
H2L5 IgG4PE antibody at 12.5 .mu.g/mL
[0038] FIG. 34: Cytokine induction of PBMC from NSCLC patients
following treatment with isotype control or H2L5 IgG4PE antibody at
10 .mu.g/mL for 72 hrs.
[0039] FIG. 35: Binding affinity of different isotype variants of
humanized H2L5 antibody to human FcgR.
[0040] FIG. 36: Binding affinity of different isotype variants
7E-17G9 to murine FcR
[0041] FIG. 37: mRNA Expression of ICOS positive cells in different
tumor pathologies from TCGA
[0042] FIG. 38: Gene expression changes with anti CD3+H2L5
treatment compared to CD3 alone in human T cells as measured by
Nanostring
SUMMARY OF THE INVENTION
[0043] In one aspect, the present invention provides methods of
treating cancer in a patient in need thereof comprising
administering to the patient an effective amount of an agent
directed to human ICOS and an effective amount of an agent directed
to human PD1 or human PD-L1 sequentially, wherein administration of
the agent directed to human ICOS is followed by administration of
the agent directed to human PD1 or human PD-L1. In one embodiment,
the agent directed to human ICOS is an ICOS agonist. In one
embodiment, the agent directed to human PD1 or human PD-L1 is a PD1
antagonist.
[0044] In one aspect, the present invention provides an anti-ICOS
antibody or antigen binding fragment thereof and an anti-PD1
antibody or antigen binding fragment thereof for sequential use in
treating cancer in a human in need thereof, wherein administration
of the anti-ICOS antibody or antigen binding fragment thereof is
followed by administration of the anti-PD1 antibody or antigen
binding fragment thereof. In one embodiment, the anti-PD1 antibody
or antigen binding fragment thereof is a PD1 antagonist. In one
embodiment, the anti-ICOS antibody or antigen binding fragment
thereof is an ICOS agonist.
[0045] In one aspect, the present invention provides an anti-ICOS
antibody or antigen binding fragment thereof and an anti-PD-L1
antibody or antigen binding fragment thereof for sequential use in
treating cancer in a human in need thereof, wherein administration
of the anti-ICOS antibody or antigen binding fragment thereof is
followed administration of the anti-PD-L1 antibody or antigen
binding fragment thereof. In one embodiment, the anti-PDL1 antibody
or antigen binding fragment thereof is a PD1 antagonist. In one
embodiment, the anti-ICOS antibody or antigen binding fragment
thereof is an ICOS agonist.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0046] As used herein "ICOS" means any Inducible T-cell
costimulator protein. Pseudonyms for ICOS (Inducible T-cell
COStimulator) include AILIM; CD278; CVID1, JTT-1 or JTT-2,
MGC39850, or 8F4. ICOS is a CD28-superfamily costimulatory molecule
that is expressed on activated T cells. The protein encoded by this
gene belongs to the CD28 and CTLA-4 cell-surface receptor family.
It forms homodimers and plays an important role in cell-cell
signaling, immune responses, and regulation of cell proliferation.
The amino acid sequence of human ICOS (isoform 2) (Accession No.:
UniProtKB-Q9Y6W8-2) is shown below as SEQ ID NO:9.
TABLE-US-00001 (SEQ ID NO: 9)
MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIV
QQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFL
YNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLP
IGCAAFVVVCILGCILICWLTKKM
[0047] The amino acid sequence of human ICOS (isoform 1) (Accession
No.: UniProtKB-Q9Y6W8-1) is shown below as SEQ ID NO: 10.
TABLE-US-00002 (SEQ ID NO: 10) MKSGLWYFFL FCLRIKVLTG EINGSANYEM
FIFHNGGVQI LCKYPDIVQQ FKMQLLKGGQ ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN
SVSFFLYNLD HSHANYYFCN LSIFDPPPFK VTLTGGYLHI YESQLCCQLK FWLPIGCAAF
VVVCILGCIL ICWLTKKKYS SSVHDPNGEY MFMRAVNTAK KSRLTDVTL
[0048] Activation of ICOS occurs through binding by ICOS-L
(B7RP-1/B7-H2). Neither B7-1 nor B7-2 (ligands for CD28 and CTLA4)
bind or activate ICOS. However, ICOS-L has been shown to bind
weakly to both CD28 and CTLA-4 (Yao S et al., "B7-H2 is a
costimulatory ligand for CD28 in human", Immunity, 34(5); 729-40
(2011)). Expression of ICOS appears to be restricted to T cells.
ICOS expression levels vary between different T cell subsets and on
T cell activation status. ICOS expression has been shown on resting
TH17, T follicular helper (TFH) and regulatory T (Treg) cells;
however, unlike CD28; it is not highly expressed on naive T.sub.H1
and T.sub.H2 effector T cell populations (Paulos C M et al., "The
inducible costimulator (ICOS) is critical for the development of
human Th17 cells", Sci Transl Med, 2(55); 55ra78 (2010)). ICOS
expression is highly induced on CD4+ and CD8+ effector T cells
following activation through TCR engagement (Wakamatsu E, et al.,
"Convergent and divergent effects of costimulatory molecules in
conventional and regulatory CD4+ T cells", Proc Natal Acad Sci USA,
110(3); 1023-8 (2013)). Co-stimulatory signalling through ICOS
receptor only occurs in T cells receiving a concurrent TCR
activation signal (Sharpe A H and Freeman G J. "The B7-CD28
Superfamily", Nat. Rev Immunol, 2(2); 116-26 (2002)). In activated
antigen specific T cells, ICOS regulates the production of both
T.sub.H1 and T.sub.H2 cytokines including IFN-.gamma., TNF-.alpha.,
IL-10, IL-4, IL-13 and others. ICOS also stimulates effector T cell
proliferation, albeit to a lesser extent than CD28 (Sharpe A H and
Freeman G J. "The B7-CD28 Superfamily", Nat. Rev Immunol, 2(2);
116-26 (2002)). Antibodies to ICOS and methods of using in the
treatment of disease are described, for instance, in WO2012/131004,
US20110243929, and US20160215059. US20160215059 is incorporated by
reference herein. CDRs for murine antibodies to human ICOS having
agonist activity are shown in PCT/EP2012/055735 (WO 2012/131004).
Antibodies to ICOS are also disclosed in WO 2008/137915, WO
2010/056804, EP 1374902, EP1374901, and EP1125585. Agonist
antibodies to ICOS or ICOS binding proteins are disclosed in
WO2012/13004, WO2014/033327, WO2016/120789, US20160215059, and
US20160304610. Exemplary antibodies in US2016/0304610 include
37A10S713. Sequences of 37A10S713 are reproduced below as SEQ ID
NOS: 14-21.
TABLE-US-00003 37A10S713 heavy chain variable region: (SEQ. ID NO :
14) EVQLVESGG LVQPGGSLRL SCAASGFTFS DYWMDWVRQA PGKGLVWVSN
IDEDGSITEY SPFVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCTRWG RFGFDSWGQG
TLVTVSS 37A10S713 light chain variable region: (SEQ. ID NO: 15)
DIVMTQSPDS LAVSLGERAT INCKSSQSLL SGSFNYLTWY QQKPGQPPKL LIFYASTRHT
GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCHHHYNAPP TFGPGTKVDI K 37A10S713
V.sub.H CDR1: (SEQ. ID NO: 16) GFTFSDYWMD 37A10S713 V.sub.H CDR2:
(SEQ. ID NO: 17) NIDEDGSITEYSPFVKG 37A10S713 V.sub.H CDR3: (SEQ.
ID. NO: 18) WGRFGFDS 37A10S713 V.sub.L CDR1: (SEQ. ID NO: 19)
KSSQSLLSGSFNYLT 37A10S713 V.sub.L CDR2: (SEQ. ID NO: 20) YASTRHT
37A10S713 V.sub.L CDR3: (SEQ. ID NO: 21) HHHYNAPPT
[0049] By "agent directed to ICOS" is meant any chemical compound
or biological molecule capable of binding to ICOS. In some
embodiments, the agent directed to ICOS is an ICOS binding protein.
In some other embodiments, the agent directed to ICOS is an ICOS
agonist.
[0050] The term "ICOS binding protein" as used herein refers to
antibodies and other protein constructs, such as domains, which are
capable of binding to ICOS. In some instances, the ICOS is human
ICOS. The term "ICOS binding protein" can be used interchangeably
with "ICOS antigen binding protein." Thus, as is understood in the
art, anti-ICOS antibodies and/or ICOS antigen binding proteins
would be considered ICOS binding proteins. As used herein, "antigen
binding protein" is any protein, including but not limited to
antibodies, domains and other constructs described herein, that
binds to an antigen, such as ICOS. As used herein "antigen binding
portion" of an ICOS binding protein would include any portion of
the ICOS binding protein capable of binding to ICOS, including but
not limited to, an antigen binding antibody fragment.
[0051] In one embodiment, the ICOS antibodies of the present
invention comprise any one or a combination of the following
CDRs:
TABLE-US-00004 CDRH1: (SEQ ID NO: 1) DYAMH CDRH2: (SEQ ID NO: 2)
LISIYSDHTNYNQKFQG CDRH3: (SEQ ID NO: 3) NNYGNYGWYFDV CDRL1: (SEQ ID
NO: 4) SASSSVSYMH CDRL2: (SEQ ID NO: 5) DTSKLAS CDRL3: (SEQ ID NO:
6) FQGSGYPYT
[0052] In some embodiments, the anti-ICOS antibodies of the present
invention comprise a heavy chain variable region having at least
90% sequence identity to SEQ ID NO:7. Suitably, the ICOS binding
proteins of the present invention may comprise a heavy chain
variable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity
to SEQ ID NO:7.
TABLE-US-00005 Humanized Heavy Chain (Vu) Variable Region (H2):
(SEQ ID NO: 7) QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYAMHWVRQA
PGQGLEWMGL ISIYSDHTNY NQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCGRNN
YGNYGWYFDV WGQGTTVTVS S
[0053] In one embodiment of the present invention the ICOS antibody
comprises CDRL1 (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), and CDRL3 (SEQ
ID NO:6) in the light chain variable region having the amino acid
sequence set forth in SEQ ID NO:8. ICOS binding proteins of the
present invention comprising the humanized light chain variable
region set forth in SEQ ID NO:8 are designated as "L5." Thus, an
ICOS binding protein of the present invention comprising the heavy
chain variable region of SEQ ID NO:7 and the light chain variable
region of SEQ ID NO:8 can be designated as H2L5 herein.
[0054] In some embodiments, the ICOS binding proteins of the
present invention comprise a light chain variable region having at
least 90% sequence identity to the amino acid sequence set forth in
SEQ ID NO:8. Suitably, the ICOS binding proteins of the present
invention may comprise a light chain variable region having about
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to SEQ ID NO:8.
TABLE-US-00006 Humanized Light Chain (VI) Variable Region (L5) (SEQ
ID NO: 8) EIVLTQSPAT LSLSPGERAT LSCSASSSVS YMHWYQQKPG QAPRLLIYDT
SKLASGIPAR FSGSGSGTDY TLTISSLEPE DFAVYYCFQG SGYPYTFGQG TKLEIK
[0055] CDRs or minimum binding units may be modified by at least
one amino acid substitution, deletion or addition, wherein the
variant antigen binding protein substantially retains the
biological characteristics of the unmodified protein, such as an
antibody comprising SEQ ID NO:7 and SEQ ID NO:8.
[0056] It will be appreciated that each of CDR H1, H2, H3, L1, L2,
L3 may be modified alone or in combination with any other CDR, in
any permutation or combination. In one embodiment, a CDR is
modified by the substitution, deletion or addition of up to 3 amino
acids, for example 1 or 2 amino acids, for example 1 amino acid.
Typically, the modification is a substitution, particularly a
conservative substitution, for example as shown in Table 1
below.
TABLE-US-00007 TABLE 1 Side chain Members Hydrophobic Met, Ala,
Val, Leu, Ile Neutral hydrophilic Cys, Ser, Thr Acidic Asp, Glu
Basic Asn, Gln, His, Lys. Arg Residues that influence chain
orientation Gly, Pro Aromatic Trp, Tyr, Phe
[0057] The subclass of an antibody in part determines secondary
effector functions, such as complement activation or Fc receptor
(FcR) binding and antibody dependent cell cytotoxicity (ADCC)
(Huber, et al., Nature 229(5284): 419-20 (1971); Brunhouse, et al.,
Mol Immunol 16(11): 907-17 (1979)). In identifying the optimal type
of antibody for a particular application, the effector functions of
the antibodies can be taken into account. For example, hIgG1
antibodies have a relatively long half life, are very effective at
fixing complement, and they bind to both Fc.gamma.RI and
Fc.gamma.RII. In contrast, human IgG4 antibodies have a shorter
half life, do not fix complement and have a lower affinity for the
FcRs. Replacement of serine 228 with a proline (S228P) in the Fc
region of IgG4 reduces heterogeneity observed with hIgG4 and
extends the serum half life (Kabat, et al., "Sequences of proteins
of immunological interest" 5.sup.th Edition (1991); Angal, et al.,
Mol Immunol 30(1): 105-8 (1993)). A second mutation that replaces
leucine 235 with a glutamic acid (L235E) eliminates the residual
FcR binding and complement binding activities (Alegre, et al., J
Immunol 148(11): 3461-8 (1992)). The resulting antibody with both
mutations is referred to as IgG4PE. The numbering of the hIgG4
amino acids was derived from EU numbering reference: Edelman, G. M.
et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969. In
one embodiment of the present invention the ICOS antibody is an
IgG4 isotype. In one embodiment, the ICOS antibody comprises an
IgG4 Fc region comprising the replacement S228P and L235E may have
the designation IgG4PE.
[0058] As used herein "ICOS-L" and "ICOS Ligand" are used
interchangeably and refer to the membrane bound natural ligand of
human ICOS. ICOS ligand is a protein that in humans is encoded by
the ICOSLG gene. ICOSLG has also been designated as CD275 (cluster
of differentiation 275). Pseudonyms for ICOS-L include B7RP-1 and
B7-H2.
[0059] As used herein, an "agent directed to PD-1" or "agent
directed to PD1" means any chemical compound or biological molecule
capable of binding to PD1. In some embodiments, the agent directed
to PD1 is a PD1 antagonist.
[0060] The term "PD1 binding protein" or "PD-1 binding protein" as
used herein refers to antibodies and other protein constructs, such
as domains, which are capable of binding to PD1. In some instances,
the PD1 is human PD1. The term "PD1 binding protein" can be used
interchangeably with "PD1 antigen binding protein." Thus, as is
understood in the art, anti-PD1 antibodies and/or PD1 antigen
binding proteins would be considered PD1 binding proteins. As used
herein, "antigen binding protein" is any protein, including but not
limited to antibodies, domains and other constructs described
herein, that binds to an antigen, such as PD1. As used herein
"antigen binding portion" of a PD1 binding protein would include
any portion of the PD1 binding protein capable of binding to PD1,
including but not limited to, an antigen binding antibody
fragment.
[0061] The protein Programmed Death 1 (PD-1) is an inhibitory
member of the CD28 family of receptors, that also includes CD28,
CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T
cells, and myeloid cells (Agata et al., supra; Okazaki et al.
(2002) Curr. Opin. Immunol 14:391779-82; Bennett et al. (2003) J
Immunol 170:711-8) The initial members of the family, CD28 and
ICOS, were discovered by functional effects on augmenting T cell
proliferation following the addition of monoclonal antibodies
(Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980)
Immunogenics 10:247-260). PD-1 was discovered through screening for
differential expression in apototic cells (Ishida et al. (1992)
EMBO J 11:3887-95) The other members of the family, CTLA-4, and
BTLA were discovered through screening for differential expression
in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS
and CTLA-4 all have an unpaired cysteine residue allowing for
homodimerization. In contrast, PD-1 is suggested to exist as a
monomer, lacking the unpaired cysteine residue characteristic in
other CD28 family members. PD-1 antibodies and methods of using in
treatment of disease are described in U.S. Pat. Nos.: U.S. Pat.
Nos. 7,595,048; 8,168,179; 8,728,474; 7,722,868; 8,008,449;
7,488,802; 7,521,051; 8,088,905; 8,168,757; 8,354,509; and US
Publication Nos. US20110171220; US20110171215; and US20110271358.
Combinations of CTLA-4 and PD-1 antibodies are described in U.S.
Pat. No. 9,084,776.
[0062] In some embodiments, the agent directed to PD1 is a PD1
antagonist and blocks binding of PD-L1 expressed on a cancer cell
to PD-1 expressed on an immune cell (T cell, B cell or NKT cell)
and may also block binding of PD-L2 expressed on a cancer cell to
the immune-cell expressed PD-1. Alternative names or synonyms for
PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1;
PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2,
PDL2, B7-DC, Btdc and CD273 for PD-L2. Human PD-1 amino acid
sequences can be found in NCBI Locus No.: NP_005009. The amino acid
sequence in NCBI Locus No.: NP_005009 is reproduced below:
TABLE-US-00008 (SEQ ID NO: 11) mqipqapwpv vwavlqlgwr pgwfldspdr
pwnpptfspa llvvtegdna tftcsfsnts esfvlnwyrm spsnqtdkla afpedrsqpg
qdcrfrvtql pngrdfhmsv vrarrndsgt ylcgaislap kaqikeslra elrvterrae
vptahpspsp rpagqfqtlv vgvvggllgs lvllvwvlav icsraargti garrtgqplk
edpsavpvfs vdygeldfqw rektpeppvp cvpeqteyat ivfpsgmgts sparrgsadg
prsaqplrpe dghcswpl
Human PD-L1 and PD-L2 amino acid sequences can be found in NCBI
Locus No.: NP_054862 and NP_079515, respectively. The amino acid
sequence in NCBI Locus No.: NP_054862 is reproduced below:
TABLE-US-00009 (SEQ ID NO: 12) mrifavfifm tywhllnaft vtvpkdlyvv
eygsnmtiec kfpvekqldl aalivyweme dkniiqfvhg eedlkvqhss yrqrarllkd
qlslgnaalq itdvklqdag vyrcmisygg adykritvkv napynkinqr ilvvdpvtse
heltcqaegy pkaeviwtss dhqvlsgktt ttnskreekl fnvtstlrin tttneifyct
frrldpeenh taelvipelp lahppnerth lvilgaillc lgvaltfifr lrkgrmmdvk
kcgiqdtnsk kqsdthleet
The amino acid sequence in NCBI Locus No.: NP_079515 is reproduced
below:
TABLE-US-00010 (SEQ ID NO: 13) miflllmlsl elqlhqiaal ftvtvpkely
iiehgsnvtl ecnfdtgshv nlgaitaslq kvendtsphr eratlleeql plgkasfhip
qvqvrdegqy qciiiygvaw dykyltlkvk asyrkinthi lkvpetdeve ltcqatgypl
aevswpnvsv pantshsrtp eglyqvtsvl rlkpppgrnf scvfwnthvr eltlasidlq
sqmeprthpt wllhifipfc iiafifiatv ialrkqlcqk lysskdttkr pvtttkrevn
sai
[0063] Agents directed to PD-1 in any of the aspects or embodiments
of the present invention include a monoclonal antibody (mAb), or
antigen binding fragment thereof, which specifically binds to PD-1.
In some embodiments, the mAb to PD-1 specifically binds to human
PD-1. The mAb may be a human antibody, a humanized antibody or a
chimeric antibody, and may include a human constant region. In some
embodiments, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG3 and IgG4 constant regions, and in
preferred embodiments, the human constant region is an IgG1 or IgG4
constant region. In some embodiments, the antigen binding fragment
is selected from the group consisting of Fab, Fab'-SH, F(ab')2,
scFv and Fv fragments.
[0064] Examples of mAbs that bind to human PD-1, and useful in the
various aspects and embodiments of the present invention, are
described in U.S. Pat. Nos. 8,552,154; 8,354,509; 8,168,757;
8,008,449; 7,521,051; 7,488,802; WO2004072286; WO2004056875; and
WO2004004771.
[0065] Other PD-1 binding proteins useful in any of the aspects and
embodiments of the present invention include an immunoadhesin that
specifically binds to PD-1, and preferably specifically binds to
human PD-1, e.g., a fusion protein containing the extracellular or
PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region
such as an Fc region of an immunoglobulin molecule. Examples of
immunoadhesin molecules that specifically bind to PD-1 are
described in WO2010027827 and WO2011066342. Specific fusion
proteins useful as the PD-1 antagonist in the treatment method,
medicaments and uses of the present invention include AMP-224 (also
known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to
human PD-1.
[0066] OPDIVO/nivolumab is a fully human monoclonal antibody
marketed by Bristol Myers Squibb directed against the negative
immunoregulatory human cell surface receptor PD-1 (programmed
death-1 or programmed cell death-1/PCD-1) with immunopotentiation
activity. Nivolumab binds to and blocks the activation of PD-1, an
Ig superfamily transmembrane protein, by its ligands PD-L1 and
PD-L2, resulting in the activation of T-cells and cell-mediated
immune responses against tumor cells or pathogens. Activated PD-1
negatively regulates T-cell activation and effector function
through the suppression of P13k/Akt pathway activation. Other names
for nivolumab include: BMS-936558, MDX-1106, and ONO-4538. The
amino acid sequence for nivolumab and methods of using and making
are disclosed in U.S. Pat. No. 8,008,449.
[0067] KEYTRUDA/pembrolizumab is an anti-PD-1 antibodies marketed
for the treatment of lung cancer by Merck. The amino acid sequence
of pembrolizumab and methods of using are disclosed in U.S. Pat.
No. 8,168,757.
[0068] By "agent directed to PD-L1" is meant any chemical compound
or biological molecule capable of binding to PD-L1. In some
embodiments, the agent directed to PD-L1 is a PD-L1 binding
protein.
[0069] The term "PDL1 binding protein" or "PD-L1 binding protein"
as used herein refers to antibodies and other protein constructs,
such as domains, which are capable of binding to PD-L1. In some
instances, the PD-L1 is human PD1. The term "PD-L1 binding protein"
can be used interchangeably with "PD-L1 antigen binding protein."
Thus, as is understood in the art, anti-PD-L1 antibodies and/or
PD-L1 antigen binding proteins would be considered PD-L1 binding
proteins. As used herein, "antigen binding protein" is any protein,
including but not limited to antibodies, domains and other
constructs described herein, that binds to an antigen, such as
PD-L1. As used herein "antigen binding portion" of a PD-L1 binding
protein would include any portion of the PD-L1 binding protein
capable of binding to PD-L1, including but not limited to, an
antigen binding antibody fragment.
[0070] In some embodiments, the agent directed to PD-L1 is a PD1
antagonist and blocks binding of PD-L1 expressed on a cancer cell
to PD-1 expressed on an immune cell (T cell, B cell or NKT cell)
and may also block binding of PD-L2 expressed on a cancer cell to
the immune-cell expressed PD-1.
[0071] PD-L1 is a B7 family member that is expressed on many cell
types, including APCs and activated T cells (Yamazaki et al. (2002)
J. Immunol. 169:5538). PD-L1 binds to both PD-1 and B7-1. Both
binding of T-cell-expressed B7-1 by PD-L1 and binding of
T-cell-expressed PD-L1 by B7-1 result in T cell inhibition (Butte
et al. (2007) Immunity 27:111). There is also evidence that, like
other B7 family members, PD-L1 can also provide costimulatory
signals to T cells (Subudhi et al. (2004) J. Clin. Invest. 113:694;
Tamura et al. (2001) Blood 97:1809). PD-L1 (human PD-L1 cDNA is
composed of the base sequence shown by EMBL/GenBank Acc. No.
AF233516 and mouse PD-L1 cDNA is composed of the base sequence
shown by NM.sub.--021893) that is a ligand of PD-1 is expressed in
so-called antigen-presenting cells (APCs) such as activated
monocytes and dendritic cells (Journal of Experimental Medicine
(2000), vol. 19, issue 7, p 1027-1034). These cells present
interaction molecules that induce a variety of immuno-inductive
signals to T lymphocytes, and PD-L1 is one of these molecules that
induce the inhibitory signal by PD-1. It has been revealed that
PD-L1 ligand stimulation suppressed the activation (cellular
proliferation and induction of various cytokine production) of PD-1
expressing T lymphocytes. PD-L1 expression has been confirmed in
not only immunocompetent cells but also a certain kind of tumor
cell lines (cell lines derived from monocytic leukemia, cell lines
derived from mast cells, cell lines derived from hepatic
carcinomas, cell lines derived from neuroblasts, and cell lines
derived from breast carcinomas) (Nature Immunology (2001), vol. 2,
issue 3, p. 261-267).
[0072] Anti-PD-L1 antibodies and methods of making the same are
known in the art. Such antibodies to PD-L1 may be polyclonal or
monoclonal, and/or recombinant, and/or humanized, and/or fully
human. PD-L1 antibodies are in development as immuno-modulatory
agents for the treatment of cancer.
[0073] Exemplary PD-L1 antibodies are disclosed in U.S. Pat. Nos.
9,212,224; 8,779,108; 8,552,154; 8,383,796; 8,217,149; US Patent
Publication No. 20110280877; WO2013079174; and WO2013019906.
Additional exemplary antibodies to PD-L1 (also referred to as CD274
or B7-H1) and methods for use are disclosed in U.S. Pat. Nos.
8,168,179; 7,943,743; 7,595,048; WO2014055897; WO2013019906; and
WO2010077634. Specific anti-human PD-L1 monoclonal antibodies
useful as a PD-1 antagonist in the treatment method, medicaments
and uses of the present invention include MPDL3280A, BMS-936559,
MEDI4736, MSB0010718C.
[0074] Atezolizumab is a fully humanized monoclonal anti-PD-L1
antibody commercially available as TECENTRIQ. Atezolizumab is
indicated for the treatment of some locally advanced or metastatic
urothelial carcinomas. Atezolizumab blocks the interaction of PD-L1
with PD-1 and CD80.
[0075] Durvalumab (previously known as MEDI4736) is a human
monoclonal antibody directed against PD-L1. Durvalumab blocks the
interaction of PD-L1 with PD-1 and CD80. Durvalumab is commercially
available as IMFINZI.TM..
[0076] Antibodies to PD-L1 (also referred to as CD274 or B7-H1) and
methods for use are disclosed in U.S. Pat. Nos. 7,943,743;
8,383,796; US20130034559, WO2014055897, U.S. Pat. Nos. 8,168,179;
and 7,595,048. PD-L1 antibodies are in development as
immuno-modulatory agents for the treatment of cancer.
[0077] As used herein the term "agonist" refers to an antigen
binding protein including but not limited to an antibody, which
upon contact with a co-signalling receptor causes one or more of
the following (1) stimulates or activates the receptor, (2)
enhances, increases or promotes, induces or prolongs an activity,
function or presence of the receptor and/or (3) enhances,
increases, promotes or induces the expression of the receptor.
Agonist activity can be measured in vitro by various assays know in
the art such as, but not limited to, measurement of cell
signalling, cell proliferation, immune cell activation markers,
cytokine production. Agonist activity can also be measured in vivo
by various assays that measure surrogate end points such as, but
not limited to the measurement of T cell proliferation or cytokine
production.
[0078] As used herein the term "antagonist" refers to an antigen
binding protein including but not limited to an antibody, which
upon contact with a co-signalling receptor causes one or more of
the following (1) attenuates, blocks or inactivates the receptor
and/or blocks activation of a receptor by its natural ligand, (2)
reduces, decreases or shortens the activity, function or presence
of the receptor and/or (3) reduces, descrease, abrogates the
expression of the receptor. Antagonist activity can be measured in
vitro by various assays know in the art such as, but not limited
to, measurement of an increase or decrease in cell signalling, cell
proliferation, immune cell activation markers, cytokine production.
Antagonist activity can also be measured in vivo by various assays
that measure surrogate end points such as, but not limited to the
measurement of T cell proliferation or cytokine production.
[0079] As used herein the term "cross competes for binding" refers
to any agent such as an antibody that will compete for binding to a
target with any of the agents of the present invention. Competition
for binding between two antibodies can be tested by various methods
known in the art including Flow cytometry, Meso Scale Discovery and
ELISA. Binding can be measured directly, meaning two or more
binding proteins can be put in contact with a co-signalling
receptor and bind may be measured for one or each. Alternatively,
binding of molecules or interest can be tested against the binding
or natural ligand and quantitatively compared with each other.
[0080] The term "binding protein" as used herein refers to
antibodies and other protein constructs, such as domains, which are
capable of binding to an antigen.
[0081] The term "antibody" is used herein in the broadest sense to
refer to molecules with an immunoglobulin-like domain (for example
IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant,
polyclonal, chimeric, human, humanized, multispecific antibodies,
including bispecific antibodies, and heteroconjugate antibodies; a
single variable domain (e.g., V.sub.H, V.sub.HH, VL, domain
antibody (dAb.TM.)), antigen binding antibody fragments, Fab,
F(ab').sub.2, Fv, disulphide linked Fv, single chain Fv,
disulphide-linked scFv, diabodies, TANDABS.TM., etc. and modified
versions of any of the foregoing.
[0082] Alternative antibody formats include alternative scaffolds
in which the one or more CDRs of the antigen binding protein can be
arranged onto a suitable non-immunoglobulin protein scaffold or
skeleton, such as an affibody, a SpA scaffold, an LDL receptor
class A domain, an avimer or an EGF domain.
[0083] The term "domain" refers to a folded protein structure which
retains its tertiary structure independent of the rest of the
protein. Generally domains are responsible for discrete functional
properties of proteins and in many cases may be added, removed or
transferred to other proteins without loss of function of the
remainder of the protein and/or of the domain.
[0084] The term "single variable domain" refers to a folded
polypeptide domain comprising sequences characteristic of antibody
variable domains. It therefore includes complete antibody variable
domains such as V.sub.H, V.sub.HH and V.sub.L and modified antibody
variable domains, for example, in which one or more loops have been
replaced by sequences which are not characteristic of antibody
variable domains, or antibody variable domains which have been
truncated or comprise N- or C-terminal extensions, as well as
folded fragments of variable domains which retain at least the
binding activity and specificity of the full-length domain. A
single variable domain is capable of binding an antigen or epitope
independently of a different variable region or domain. A "domain
antibody" or "dAb.TM." may be considered the same as a "single
variable domain". A single variable domain may be a human single
variable domain, but also includes single variable domains from
other species such as rodent nurse shark and Camelid V.sub.HH
dAbs.TM.. Camelid V.sub.HH are immunoglobulin single variable
domain polypeptides that are derived from species including camel,
llama, alpaca, dromedary, and guanaco, which produce heavy chain
antibodies naturally devoid of light chains. Such V.sub.HH domains
may be humanized according to standard techniques available in the
art, and such domains are considered to be "single variable
domains". As used herein V.sub.H includes camelid V.sub.HH
domains.
[0085] An antigen binding fragment may be provided by means of
arrangement of one or more CDRs on non-antibody protein scaffolds.
"Protein Scaffold" as used herein includes but is not limited to an
immunoglobulin (Ig) scaffold, for example an IgG scaffold, which
may be a four chain or two chain antibody, or which may comprise
only the Fc region of an antibody, or which may comprise one or
more constant regions from an antibody, which constant regions may
be of human or primate origin, or which may be an artificial
chimera of human and primate constant regions.
[0086] The protein scaffold may be an Ig scaffold, for example an
IgG, or IgA scaffold. The IgG scaffold may comprise some or all the
domains of an antibody (i.e. CH1, CH2, CH3, V.sub.H, V.sub.L). The
antigen binding protein may comprise an IgG scaffold selected from
IgG1, IgG2, IgG3, IgG4 or IgG4PE. For example, the scaffold may be
IgG1. The scaffold may consist of, or comprise, the Fc region of an
antibody, or is a part thereof.
[0087] Affinity is the strength of binding of one molecule, e.g. an
antigen binding protein of the invention, to another, e.g. its
target antigen, at a single binding site. The binding affinity of
an antigen binding protein to its target may be determined by
equilibrium methods (e.g. enzyme-linked immunoabsorbent assay
(ELISA) or radioimmunoassay (RIA)), or kinetics (e.g. BIACORE.TM.
analysis). For example, the BIACORE.TM. methods described in
Example 5 may be used to measure binding affinity.
[0088] Avidity is the sum total of the strength of binding of two
molecules to one another at multiple sites, e.g. taking into
account the valency of the interaction.
[0089] By "isolated" it is intended that the molecule, such as an
antigen binding protein or nucleic acid, is removed from the
environment in which it may be found in nature. For example, the
molecule may be purified away from substances with which it would
normally exist in nature. For example, the mass of the molecule in
a sample may be 95% of the total mass.
[0090] The term "expression vector" as used herein means an
isolated nucleic acid which can be used to introduce a nucleic acid
of interest into a cell, such as a eukaryotic cell or prokaryotic
cell, or a cell free expression system where the nucleic acid
sequence of interest is expressed as a peptide chain such as a
protein. Such expression vectors may be, for example, cosmids,
plasmids, viral sequences, transposons, and linear nucleic acids
comprising a nucleic acid of interest. Once the expression vector
is introduced into a cell or cell free expression system (e.g.,
reticulocyte lysate) the protein encoded by the nucleic acid of
interest is produced by the transcription/translation machinery.
Expression vectors within the scope of the disclosure may provide
necessary elements for eukaryotic or prokaryotic expression and
include viral promoter driven vectors, such as CMV promoter driven
vectors, e.g., pcDNA3.1, pCEP4, and their derivatives, Baculovirus
expression vectors, Drosophila expression vectors, and expression
vectors that are driven by mammalian gene promoters, such as human
Ig gene promoters. Other examples include prokaryotic expression
vectors, such as T7 promoter driven vectors, e.g., pET41, lactose
promoter driven vectors and arabinose gene promoter driven vectors.
Those of ordinary skill in the art will recognize many other
suitable expression vectors and expression systems.
[0091] The term "recombinant host cell" as used herein means a cell
that comprises a nucleic acid sequence of interest that was
isolated prior to its introduction into the cell. For example, the
nucleic acid sequence of interest may be in an expression vector
while the cell may be prokaryotic or eukaryotic. Exemplary
eukaryotic cells are mammalian cells, such as but not limited to,
COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2/0, NS0,
293, HeLa, myeloma, lymphoma cells or any derivative thereof. Most
preferably, the eukaryotic cell is a HEK293, NS0, SP2/0, or CHO
cell. E. coli is an exemplary prokaryotic cell. A recombinant cell
according to the disclosure may be generated by transfection, cell
fusion, immortalization, or other procedures well known in the art.
A nucleic acid sequence of interest, such as an expression vector,
transfected into a cell may be extrachromasomal or stably
integrated into the chromosome of the cell.
[0092] A "chimeric antibody" refers to a type of engineered
antibody which contains a naturally-occurring variable region
(light chain and heavy chains) derived from a donor antibody in
association with light and heavy chain constant regions derived
from an acceptor antibody.
[0093] A "humanized antibody" refers to a type of engineered
antibody having its CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one or more human immunoglobulin(s). In
addition, framework support residues may be altered to preserve
binding affinity (see, e.g., Queen et al. Proc. Natl Acad Sci USA,
86:10029-10032 (1989), Hodgson, et al., Bio/Technology, 9:421
(1991)). A suitable human acceptor antibody may be one selected
from a conventional database, e.g., the KABAT.TM. database, Los
Alamos database, and Swiss Protein database, by homology to the
nucleotide and amino acid sequences of the donor antibody. A human
antibody characterized by a homology to the framework regions of
the donor antibody (on an amino acid basis) may be suitable to
provide a heavy chain constant region and/or a heavy chain variable
framework region for insertion of the donor CDRs. A suitable
acceptor antibody capable of donating light chain constant or
variable framework regions may be selected in a similar manner. It
should be noted that the acceptor antibody heavy and light chains
are not required to originate from the same acceptor antibody. The
prior art describes several ways of producing such humanized
antibodies--see, for example, EP-A-0239400 and EP-A-054951.
[0094] The term "fully human antibody" includes antibodies having
variable and constant regions (if present) derived from human
germline immunoglobulin sequences. The human sequence antibodies of
the invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). Fully human antibodies comprise amino acid sequences
encoded only by polynucleotides that are ultimately of human origin
or amino acid sequences that are identical to such sequences. As
meant herein, antibodies encoded by human immunoglobulin-encoding
DNA inserted into a mouse genome produced in a transgenic mouse are
fully human antibodies since they are encoded by DNA that is
ultimately of human origin. In this situation, human
immunoglobulin-encoding DNA can be rearranged (to encode an
antibody) within the mouse, and somatic mutations may also occur.
Antibodies encoded by originally human DNA that has undergone such
changes in a mouse are fully human antibodies as meant herein. The
use of such transgenic mice makes it possible to select fully human
antibodies against a human antigen. As is understood in the art,
fully human antibodies can be made using phage display technology
wherein a human DNA library is inserted in phage for generation of
antibodies comprising human germline DNA sequence.
[0095] The term "donor antibody" refers to an antibody that
contributes the amino acid sequences of its variable regions, CDRs,
or other functional fragments or analogs thereof to a first
immunoglobulin partner. The donor, therefore, provides the altered
immunoglobulin coding region and resulting expressed altered
antibody with the antigenic specificity and neutralising activity
characteristic of the donor antibody.
[0096] The term "acceptor antibody" refers to an antibody that is
heterologous to the donor antibody, which contributes all (or any
portion) of the amino acid sequences encoding its heavy and/or
light chain framework regions and/or its heavy and/or light chain
constant regions to the first immunoglobulin partner. A human
antibody may be the acceptor antibody.
[0097] The terms "V.sub.H" and "V.sub.L" are used herein to refer
to the heavy chain variable region and light chain variable region
respectively of an antigen binding protein.
[0098] "CDRs" are defined as the complementarity determining region
amino acid sequences of an antigen binding protein. These are the
hypervariable regions of immunoglobulin heavy and light chains.
There are three heavy chain and three light chain CDRs (or CDR
regions) in the variable portion of an immunoglobulin. Thus, "CDRs"
as used herein refers to all three heavy chain CDRs, all three
light chain CDRs, all heavy and light chain CDRs, or at least two
CDRs.
[0099] Throughout this specification, amino acid residues in
variable domain sequences and full length antibody sequences are
numbered according to the Kabat numbering convention. Similarly,
the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2",
"CDRH3" used in the Examples follow the Kabat numbering convention.
For further information, see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed., U.S. Department of Health and
Human Services, National Institutes of Health (1991).
[0100] It will be apparent to those skilled in the art that there
are alternative numbering conventions for amino acid residues in
variable domain sequences and full length antibody sequences. There
are also alternative numbering conventions for CDR sequences, for
example those set out in Chothia et al. (1989) Nature 342: 877-883.
The structure and protein folding of the antibody may mean that
other residues are considered part of the CDR sequence and would be
understood to be so by a skilled person.
[0101] Other numbering conventions for CDR sequences available to a
skilled person include "AbM" (University of Bath) and "contact"
(University College London) methods. The minimum overlapping region
using at least two of the Kabat, Chothia, AbM and contact methods
can be determined to provide the "minimum binding unit". The
minimum binding unit may be a sub-portion of a CDR.
[0102] "Percent identity" between a query nucleic acid sequence and
a subject nucleic acid sequence is the "Identities" value,
expressed as a percentage, that is calculated by the BLASTN
algorithm when a subject nucleic acid sequence has 100% query
coverage with a query nucleic acid sequence after a pair-wise
BLASTN alignment is performed. Such pair-wise BLASTN alignments
between a query nucleic acid sequence and a subject nucleic acid
sequence are performed by using the default settings of the BLASTN
algorithm available on the National Center for Biotechnology
Institute's website with the filter for low complexity regions
turned off.
[0103] "Percent identity" between a query amino acid sequence and a
subject amino acid sequence is the "Identities" value, expressed as
a percentage, that is calculated by the BLASTP algorithm when a
subject amino acid sequence has 100% query coverage with a query
amino acid sequence after a pair-wise BLASTP alignment is
performed. Such pair-wise BLASTP alignments between a query amino
acid sequence and a subject amino acid sequence are performed by
using the default settings of the BLASTP algorithm available on the
National Center for Biotechnology Institute's website with the
filter for low complexity regions turned off.
[0104] The query sequence may be 100% identical to the subject
sequence, or it may include up to a certain integer number of amino
acid or nucleotide alterations as compared to the subject sequence
such that the % identity is less than 100%. For example, the query
sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or
99% identical to the subject sequence. Such alterations include at
least one amino acid deletion, substitution (including conservative
and non-conservative substitution), or insertion, and wherein said
alterations may occur at the amino- or carboxy-terminal positions
of the query sequence or anywhere between those terminal positions,
interspersed either individually among the amino acids or
nucleotides in the query sequence or in one or more contiguous
groups within the query sequence.
[0105] The % identity may be determined across the entire length of
the query sequence, including the CDR(s). Alternatively, the %
identity may exclude the CDR(s), for example the CDR(s) is 100%
identical to the subject sequence and the % identity variation is
in the remaining portion of the query sequence, so that the CDR
sequence is fixed/intact.
[0106] In one aspect, methods of treating cancer in a patient in
need thereof, comprising administering to the patient an effective
amount of an agent directed to human ICOS and an effective amount
of an agent directed to human PD1 or human PD-L1 sequentially are
provided. In one embodiment, administration of the agent directed
to human ICOS is followed by administration of the agent directed
to human PD1 or human PD-L1. In one embodiment, the agent directed
to human PD1 or human PD-L1 is administered concurrently with an
agent directed to human ICOS in the phase following administration
of the agent directed to human ICOS.
[0107] In another aspect, administration of the agent directed to
human PD1 or human PD-L1 is followed by administration of the agent
directed to human ICOS. In one embodiment, the agent directed to
human ICOS is an anti-ICOS antibody or antigen binding portion
thereof. In one embodiment, the agent directed to human ICOS is
administered concurrently with an agent directed to human PD1 or
human PD-L1 in the phase following administration of the agent
directed to human PD1 or human PD-L1.
[0108] In one aspect, an anti-ICOS antibody or antigen binding
fragment thereof and an anti-PD 1 antibody or antigen binding
fragment thereof for sequential use in treating cancer in a human
in need thereof are provided. In one embodiment, administration of
the anti-ICOS antibody or antigen binding fragment thereof is
followed by administration of the anti-PD1 antibody or antigen
binding fragment thereof. In another embodiment, administration of
the anti-PD1 antibody or antigen binding fragment thereof is
followed by administration of the anti-ICOS antibody or antigen
binding fragment thereof.
[0109] In one aspect, an anti-ICOS antibody or antigen binding
fragment thereof and an anti-PD-L1 antibody or antigen binding
fragment thereof for sequential use in treating cancer in a human
in need thereof are provided. In one embodiment, administration of
the anti-ICOS antibody or antigen binding fragment thereof is
followed by administration of the anti-PD-L1 antibody or antigen
binding fragment thereof. In another embodiment, administration of
the anti-PD-L1 antibody or antigen binding fragment thereof is
followed by administration of the anti-ICOS antibody or antigen
binding fragment thereof.
[0110] In another aspect, use of an anti-ICOS antibody or antigen
binding portion thereof and an anti-PD1 antibody or antigen binding
portion thereof in the manufacture of a medicament for the
treatment of cancer is provided, wherein the anti-ICOS antibody or
antigen binding portion thereof and an anti-PD1 antibody or antigen
binding portion thereof are sequentially administered, and wherein
administration of the anti-ICOS antibody or antigen binding portion
thereof is followed by administration of the anti-PD1 antibody or
antigen binding portion thereof.
[0111] In another aspect, use of an anti-ICOS antibody or antigen
binding portion thereof and an anti-PDL1 antibody or antigen
binding portion thereof in the manufacture of a medicament for the
treatment of cancer is provided, wherein the anti-ICOS antibody or
antigen binding portion thereof and an anti-PDL1 antibody or
antigen binding portion thereof are sequentially administered, and
wherein administration of the anti-ICOS antibody or antigen binding
portion thereof is followed by administration of the anti-PDL1
antibody or antigen binding portion thereof.
[0112] The present invention also provides polynucleotides encoding
anti-ICOS antibodies, anti-PD1 antibodies, anti-PDL1 antibodies, or
antigen binding portion of any one of said antibodies, of the
present invention. In one embodiment, host cells are provided
comprising polynucleotides encoding anti-ICOS antibodies, anti-PD1
antibodies, or anti-PDL1 antibodies, or antigen binding portions of
any one of said antibodies, of the present invention. The present
invention also provides methods of making an anti-ICOS antibody,
anti-PD1 antibody, anti-PDL1 antibody, or an antigen binding
portion of said antibody, comprising the steps of a) culturing host
cell comprising a polynucleotide encoding an anti-ICOS antibody,
anti-PD1 antibody, or anti-PDL1 antibody or an antigen binding
portion of said antibody of the present invention under suitable
conditions to express said anti-ICOS antibody, anti-PD1 antibody,
or anti-PDL1 antibody or antigen binding portion of said antibody;
and b) isolating said anti-ICOS, anti-PD1, or anti-PDL1 antibody or
antigen binding portion of said antibody.
[0113] In another aspect, a polynucleotide encoding an anti-ICOS
antibody or antigen binding portion thereof is provided, wherein
the anti-ICOS antibody or antigen binding portion thereof is
sequentially administered to a cancer patient with an anti-PD1
antibody or antigen binding portion thereof, and wherein
administration of the anti-ICOS antibody or antigen binding portion
thereof is followed by administration of the anti-PD1 antibody or
antigen binding portion thereof.
[0114] In another aspect, a polynucleotide encoding an anti-ICOS
antibody or antigen binding portion thereof is provided, wherein
the anti-ICOS antibody or antigen binding portion thereof is
sequentially administered to a cancer patient with an anti-PDL1
antibody or antigen binding portion thereof, and wherein
administration of the anti-ICOS antibody or antigen binding portion
thereof is followed by administration of the anti-PDL1 antibody or
antigen binding portion thereof.
[0115] In yet another aspect, a polynucleotide encoding an anti-PD1
antibody or antigen binding portion thereof is provided, wherein
the anti-PD1 antibody or antigen binding portion thereof is
sequentially administered to a cancer patient with an anti-ICOS
antibody or antigen binding portion thereof, and wherein
administration of the anti-ICOS antibody or antigen binding portion
thereof is followed by administration of the anti-PD1 antibody or
antigen binding portion thereof.
[0116] In still another aspect, a polynucleotide encoding an
anti-PDL1 antibody or antigen binding portion thereof is provided,
wherein the anti-PDL1 antibody or antigen binding portion thereof
is sequentially administered to a cancer patient with an anti-ICOS
antibody or antigen binding portion thereof, and wherein
administration of the anti-ICOS antibody or antigen binding portion
thereof is followed by administration of the anti-PDL1 antibody or
antigen binding portion thereof.
[0117] In another aspect, a vector comprising the polynucleotide of
any one of the aspects herein is provided. In another aspect, a
host cell comprising the vector of any one of the aspects herein is
provided.
[0118] In yet another aspect, a method of making an anti-ICOS
antibody or antigen binding portion thereof is provided, the method
comprising a) culturing a host cell comprising the polynucleotide
of any one of the aspects herein under suitable conditions to
express the anti-ICOS antibody or antigen binding portion thereof;
and b) isolating said anti-ICOS antibody or antigen binding portion
thereof.
[0119] In another aspect, a method of making an anti-PD1 antibody
or antigen binding portion thereof is provided, the method
comprising a) culturing a host cell comprising the polynucleotide
of any one of the aspects herein under suitable conditions to
express the anti-PD 1 antibody or antigen binding portion thereof,
and b) isolating said anti-PD1 antibody or antigen binding portion
thereof.
[0120] In still another aspect, a method of making an anti-PDL1
antibody or antigen binding portion thereof is provided, the method
comprising a) culturing a host cell comprising the polynucleotide
of any one of the aspects herein under suitable conditions to
express the anti-PDL1 antibody or antigen binding portion thereof,
and b) isolating said anti-PDL1 antibody or antigen binding portion
thereof.
[0121] In one embodiment of any one of the aspects herein, the
anti-ICOS antibody is an ICOS agonist. In one embodiment, the
anti-ICOS antibody comprises a V.sub.H domain comprising an amino
acid sequence at least 90% identical to the amino acid sequence set
forth in SEQ ID NO:7; and a V.sub.L domain comprising an amino acid
sequence at least 90% identical to the amino acid sequence as set
forth in SEQ ID NO:8. In another embodiment, the anti-ICOS antibody
comprises a V.sub.H domain comprising the amino acid sequence set
forth in SEQ ID NO:7 and a V.sub.L domain comprising the amino acid
sequence as set forth in SEQ ID NO:8. In one embodiment, the
anti-ICOS antibody comprises one or more of: CDRH1 as set forth in
SEQ ID NO: 1; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth
in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set
forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ ID NO:6 or a
direct equivalent of each CDR wherein a direct equivalent has no
more than two amino acid substitutions in said CDR.
[0122] In one embodiment of any one of the aspects herein, the
agent directed to human PD1 is an anti-PD1 antibody or antigen
binding portion thereof. In one embodiment, the anti-PD1 antibody
is a PD1 antagonist. In one embodiment, the anti-PD1 antibody is
pembrolizumab. In another embodiment, the anti-PD1 antibody is
nivolumab. In one embodiment of any one of the aspects herein, the
agent directed to human PD-L1 is an anti-PD-L1 antibody or antigen
binding portion thereof. In one embodiment, the anti-PD-L1 antibody
is a PD1 antagonist. In one embodiment, the anti-PD-L1 antibody is
durvalumab.
[0123] In one embodiment of any one of the aspects herein, the
agent directed to human ICOS is administered for 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30 consecutive days. In one embodiment of
any one of the aspects herein, the agent directed to human PD1 or
human PD-L1 is administered for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30 consecutive days.
[0124] In one aspect, the cancer is selected from the group
consisting of colorectal cancer (CRC), gastric, esophageal,
cervical, bladder, breast, head and neck, ovarian, melanoma, renal
cell carcinoma (RCC), EC squamous cell, non-small cell lung
carcinoma, mesothelioma, pancreatic, and prostate cancer.
[0125] In one aspect, the present invention provides a method of
treating cancer in a human in need thereof, the method comprising
administering to said human an anti-ICOS antibody or antigen
binding fragment thereof and/or administering to said human an
anti-PD1 antibody or antigen binding fragment thereof. In one
embodiment, the anti-ICOS antibody or antigen binding fragment
thereof induces T-cell proliferation, expansion, and tumor
infiltration. In another embodiment, the anti-ICOS antibody or
antigen binding fragment thereof increases PD-1 expression on a
T-cell. In one embodiment, the anti-PD1 antibody or antigen binding
fragment thereof increases ICOS expression on a T-cell. In one
embodiment, the anti-ICOS antibody or antigen binding fragment
thereof is an IgG4 isotype and reduces depletion of ICOS-positive
T-cells. In another embodiment, the anti-ICOS antibody or antigen
binding fragment thereof is an IgG4 isotype and results in
increased anti-cancer efficacy when compared to an IgG1 isotype
anti-ICOS antibody.
[0126] In another embodiment the cancer is selected from head and
neck cancer, breast cancer, lung cancer, colon cancer, ovarian
cancer, prostate cancer, gliomas, glioblastoma, astrocytomas,
glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease,
Lhermitte-Duclos disease, inflammatory breast cancer, Wilm's tumor,
Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma,
kidney cancer, liver cancer, melanoma, pancreatic cancer, sarcoma,
osteosarcoma, giant cell tumor of bone, thyroid cancer,
lymphoblastic T cell leukemia, Chronic myelogenous leukemia,
Chronic lymphocytic leukemia, Hairy-cell leukemia, acute
lymphoblastic leukemia, acute myelogenous leukemia, AML, Chronic
neutrophilic leukemia, Acute lymphoblastic T cell leukemia,
plasmacytoma, Immunoblastic large cell leukemia, Mantle cell
leukemia, Multiple myeloma Megakaryoblastic leukemia, multiple
myeloma, acute megakaryocytic leukemia, promyelocytic leukemia,
Erythroleukemia, malignant lymphoma, hodgkins lymphoma,
non-hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt's
lymphoma, follicular lymphoma, neuroblastoma, bladder cancer,
urothelial cancer, vulval cancer, cervical cancer, endometrial
cancer, renal cancer, mesothelioma, esophageal cancer, salivary
gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal
cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal
stromal tumor), and testicular cancer.
[0127] Some embodiments of the present invention further comprise
administering at least one neo-plastic agent and/or at least one
immunostimulatory agent to said human.
[0128] In one aspect the human has a solid tumor. In one aspect the
tumor is selected from head and neck cancer, gastric cancer,
melanoma, renal cell carcinoma (RCC), esophageal cancer, non-small
cell lung carcinoma, prostate cancer, colorectal cancer, ovarian
cancer and pancreatic cancer. In another aspect the human has a
liquid tumor such as diffuse large B cell lymphoma (DLBCL),
multiple myeloma, chronic lyphomblastic leukemia (CLL), follicular
lymphoma, acute myeloid leukemia and chronic myelogenous
leukemia.
[0129] The present disclosure also relates to a method for treating
or lessening the severity of a cancer selected from: brain
(gliomas), glioblastomas, Bannayan-Zonana syndrome, Cowden disease,
Lhermitte-Duclos disease, breast, inflammatory breast cancer,
Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,
medulloblastoma, colon, head and neck, kidney, lung, liver,
melanoma, ovarian, pancreatic, prostate, sarcoma, osteosarcoma,
giant cell tumor of bone, thyroid, lymphoblastic T-cell leukemia,
chronic myelogenous leukemia, chronic lymphocytic leukemia,
hairy-cell leukemia, acute lymphoblastic leukemia, acute
myelogenous leukemia, chronic neutrophilic leukemia, acute
lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large
cell leukemia, mantle cell leukemia, multiple myeloma
megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic
leukemia, promyelocytic leukemia, erythroleukemia, malignant
lymphoma, Hodgkins lymphoma, non-hodgkins lymphoma, lymphoblastic T
cell lymphoma, Burkitt's lymphoma, follicular lymphoma,
neuroblastoma, bladder cancer, urothelial cancer, lung cancer,
vulval cancer, cervical cancer, endometrial cancer, renal cancer,
mesothelioma, esophageal cancer, salivary gland cancer,
hepatocellular cancer, gastric cancer, nasopharangeal cancer,
buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal
tumor) and testicular cancer.
[0130] By the term "treating" and grammatical variations thereof as
used herein, is meant therapeutic therapy. In reference to a
particular condition, treating means: (1) to ameliorate the
condition or one or more of the biological manifestations of the
condition, (2) to interfere with (a) one or more points in the
biological cascade that leads to or is responsible for the
condition or (b) one or more of the biological manifestations of
the condition, (3) to alleviate one or more of the symptoms,
effects or side effects associated with the condition or treatment
thereof, or (4) to slow the progression of the condition or one or
more of the biological manifestations of the condition.
Prophylactic therapy using the methods and/or compositions of the
invention is also contemplated. The skilled artisan will appreciate
that "prevention" is not an absolute term. In medicine,
"prevention" is understood to refer to the prophylactic
administration of a drug to substantially diminish the likelihood
or severity of a condition or biological manifestation thereof, or
to delay the onset of such condition or biological manifestation
thereof. Prophylactic therapy is appropriate, for example, when a
subject is considered at high risk for developing cancer, such as
when a subject has a strong family history of cancer or when a
subject has been exposed to a carcinogen.
[0131] As used herein, the terms "cancer," "neoplasm," and "tumor"
are used interchangeably and, in either the singular or plural
form, refer to cells that have undergone a malignant transformation
that makes them pathological to the host organism. Primary cancer
cells can be readily distinguished from non-cancerous cells by
well-established techniques, particularly histological examination.
The definition of a cancer cell, as used herein, includes not only
a primary cancer cell, but any cell derived from a cancer cell
ancestor. This includes metastasized cancer cells, and in vitro
cultures and cell lines derived from cancer cells. When referring
to a type of cancer that normally manifests as a solid tumor, a
"clinically detectable" tumor is one that is detectable on the
basis of tumor mass; e.g., by procedures such as computed
tomography (CT) scan, magnetic resonance imaging (MRI), X-ray,
ultrasound or palpation on physical examination, and/or which is
detectable because of the expression of one or more cancer-specific
antigens in a sample obtainable from a patient. Tumors may be a
hematopoietic (or hematologic or hematological or blood-related)
cancer, for example, cancers derived from blood cells or immune
cells, which may be referred to as "liquid tumors." Specific
examples of clinical conditions based on hematologic tumors include
leukemias such as chronic myelocytic leukemia, acute myelocytic
leukemia, chronic lymphocytic leukemia and acute lymphocytic
leukemia; plasma cell malignancies such as multiple myeloma, MGUS
and Waldenstrom's macroglobulinemia; lymphomas such as
non-Hodgkin's lymphoma, Hodgkin's lymphoma; and the like.
[0132] The cancer may be any cancer in which an abnormal number of
blast cells or unwanted cell proliferation is present or that is
diagnosed as a hematological cancer, including both lymphoid and
myeloid malignancies. Myeloid malignancies include, but are not
limited to, acute myeloid (or myelocytic or myelogenous or
myeloblastic) leukemia (undifferentiated or differentiated), acute
promyeloid (or promyelocytic or promyelogenous or promyeloblastic)
leukemia, acute myelomonocytic (or myelomonoblastic) leukemia,
acute monocytic (or monoblastic) leukemia, erythroleukemia and
megakaryocytic (or megakaryoblastic) leukemia. These leukemias may
be referred together as acute myeloid (or myelocytic or
myelogenous) leukemia (AML). Myeloid malignancies also include
myeloproliferative disorders (MPD) which include, but are not
limited to, chronic myelogenous (or myeloid) leukemia (CML),
chronic myelomonocytic leukemia (CMML), essential thrombocythemia
(or thrombocytosis), and polcythemia vera (PCV). Myeloid
malignancies also include myelodysplasia (or myelodysplastic
syndrome or MDS), which may be referred to as refractory anemia
(RA), refractory anemia with excess blasts (RAEB), and refractory
anemia with excess blasts in transformation (RAEBT); as well as
myelofibrosis (MFS) with or without agnogenic myeloid
metaplasia.
[0133] Hematopoietic cancers also include lymphoid malignancies,
which may affect the lymph nodes, spleens, bone marrow, peripheral
blood, and/or extranodal sites. Lymphoid cancers include B-cell
malignancies, which include, but are not limited to, B-cell
non-Hodgkin's lymphomas (B-NHLs). B-NHLs may be indolent (or
low-grade), intermediate-grade (or aggressive) or high-grade (very
aggressive). Indolent Bcell lymphomas include follicular lymphoma
(FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma
(MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic
MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and
mucosa-associated-lymphoid tissue (MALT or extranodal marginal
zone) lymphoma. Intermediate-grade B-NHLs include mantle cell
lymphoma (MCL) with or without leukemic involvement, diffuse large
cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade
3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade
B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma,
small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma.
Other B-NHLs include immunoblastic lymphoma (or immunocytoma),
primary effusion lymphoma, HIV associated (or AIDS related)
lymphomas, and post-transplant lymphoproliferative disorder (PTLD)
or lymphoma. B-cell malignancies also include, but are not limited
to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia
(PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia
(HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or
lymphocytic or lymphoblastic) leukemia, and Castleman's disease.
NHL may also include T-cell non-Hodgkin's lymphoma s(T-NHLs), which
include, but are not limited to T-cell non-Hodgkin's lymphoma not
otherwise specified (NOS), peripheral T-cell lymphoma (PTCL),
anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid
disorder (AILD), nasal natural killer (NK) cell/T-cell lymphoma,
gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides,
and Sezary syndrome.
[0134] Hematopoietic cancers also include Hodgkin's lymphoma (or
disease) including classical Hodgkin's lymphoma, nodular sclerosing
Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma,
lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP
Hodgkin's lymphoma, and lymphocyte depleted Hodgkin's lymphoma.
Hematopoietic cancers also include plasma cell diseases or cancers
such as multiple myeloma (MM) including smoldering MM, monoclonal
gammopathy of undetermined (or unknown or unclear) significance
(MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic
lymphoma (LPL), Waldenstrom's Macroglobulinemia, plasma cell
leukemia, and primary amyloidosis (AL). Hematopoietic cancers may
also include other cancers of additional hematopoietic cells,
including polymorphonuclear leukocytes (or neutrophils), basophils,
eosinophils, dendritic cells, platelets, erythrocytes and natural
killer cells. Tissues which include hematopoietic cells referred
herein to as "hematopoietic cell tissues" include bone marrow;
peripheral blood; thymus; and peripheral lymphoid tissues, such as
spleen, lymph nodes, lymphoid tissues associated with mucosa (such
as the gut-associated lymphoid tissues), tonsils, Peyer's patches
and appendix, and lymphoid tissues associated with other mucosa,
for example, the bronchial linings.
[0135] As used herein the term "Compound A.sup.2" means an agent
directed to human ICOS. In some embodiments, Compound A.sup.2 is an
antibody to human ICOS or the antigen binding 5 portion thereof. In
some embodiments, Compound A.sup.2 is an ICOS agonist. Suitably
Compound A.sup.2 means a humanized monoclonal antibody having a
heavy chain variable region as set forth in SEQ ID NO:7 and a light
chain variable region as set forth in SEQ ID NO:8.
[0136] As used herein the term "Compound B.sup.2" means an agent
directed to human PD1 or an agent to directed to human PD-L1. In
some embodiments, Compound B.sup.2 is a PD1 antagonist. In some
embodiments, Compound B.sup.2 is an antibody to human PD1 or the
antigen binding portion thereof. In some embodiments, Compound
B.sup.2 is an antibody to human PD-L1 or the antigen binding
portion thereof. Suitably, Compound B.sup.2 is nivolumab. Suitably,
Compound B.sup.2 is pembrolizumab.
[0137] Suitably, the combinations of this invention are
administered within a "specified period".
[0138] The term "specified period" and grammatical variations
thereof, as used herein, means the interval of time between the
administration of one of Compound A.sup.2 and Compound B.sup.2 and
the other of Compound A.sup.2 and Compound B.sup.2.
[0139] Suitably, if the compounds are administered within a
"specified period" and not administered simultaneously, they are
both administered within about 24 hours of each other--in this
case, the specified period will be about 24 hours; suitably they
will both be administered within about 12 hours of each other--in
this case, the specified period will be about 12 hours; suitably
they will both be administered within about 11 hours of each
other--in this case, the specified period will be about 11 hours;
suitably they will both be administered within about 10 hours of
each other--in this case, the specified period will be about 10
hours; suitably they will both be administered within about 9 hours
of each other--in this case, the specified period will be about 9
hours; suitably they will both be administered within about 8 hours
of each other--in this case, the specified period will be about 8
hours; suitably they will both be administered within about 7 hours
of each other--in this case, the specified period will be about 7
hours; suitably they will both be administered within about 6 hours
of each other--in this case, the specified period will be about 6
hours; suitably they will both be administered within about 5 hours
of each other--in this case, the specified period will be about 5
hours; suitably they will both be administered within about 4 hours
of each other--in this case, the specified period will be about 4
hours; suitably they will both be administered within about 3 hours
of each other--in this case, the specified period will be about 3
hours; suitably they will be administered within about 2 hours of
each other--in this case, the specified period will be about 2
hours; suitably they will both be administered within about 1 hour
of each other--in this case, the specified period will be about 1
hour. As used herein, the administration of Compound A.sup.2 and
Compound B.sup.2 in less than about 45 minutes apart is considered
simultaneous administration.
[0140] Suitably, when the combination of the invention is
administered for a "specified period", the compounds will be
co-administered for a "duration of time".
[0141] The term "duration of time" and grammatical variations
thereof, as used herein means that both compounds of the invention
are administered for an indicated number of consecutive days.
Unless otherwise defined, the number of consecutive days does not
have to commence with the start of treatment or terminate with the
end of treatment, it is only required that the number of
consecutive days occur at some point during the course of
treatment.
Regarding "Specified Period" Administration:
[0142] Suitably, both compounds will be administered within a
specified period for at least one day--in this case, the duration
of time will be at least one day; suitably, during the course to
treatment, both compounds will be administered within a specified
period for at least 3 consecutive days--in this case, the duration
of time will be at least 3 days; suitably, during the course to
treatment, both compounds will be administered within a specified
period for at least 5 consecutive days--in this case, the duration
of time will be at least 5 days; suitably, during the course to
treatment, both compounds will be administered within a specified
period for at least 7 consecutive days--in this case, the duration
of time will be at least 7 days; suitably, during the course to
treatment, both compounds will be administered within a specified
period for at least 14 consecutive days--in this case, the duration
of time will be at least 14 days; suitably, during the course to
treatment, both compounds will be administered within a specified
period for at least 30 consecutive days--in this case, the duration
of time will be at least 30 days.
[0143] Suitably, if the compounds are not administered during a
"specified period", they are administered sequentially. By the term
"sequential administration", and grammatical derivates thereof, as
used herein is meant that one of Compound A.sup.2 and Compound
B.sup.2 is administered for two or more consecutive days and the
other of Compound A.sup.2 and Compound B.sup.2 is subsequently
administered for two or more consecutive days. During the period of
consecutive days in which Compound A.sup.2 is administered, at
least 1 dose, at least 2 doses, at least 3 doses, at least 4 doses,
at least 5 doses, at least 6 doses, at least 7 doses, at least 8
doses, at least 9 doses, or at least 10 doses of Compound A.sup.2
is administered. During the period of consecutive days in which
Compound B.sup.2 is administered, at least 1 dose, at least 2
doses, at least 3 doses, at least 4 doses, at least 5 doses, at
least 6 doses, at least 7 doses, at least 8 doses, at least 9
doses, or at least 10 doses Compound B.sup.2 is administered.
During the period of consecutive days in which Compound A.sup.2 is
administered, Compound A.sup.2 can be administered at least three
times a day, at least twice a day, at least once a day, or less
than once a day, e.g., once every 2 days, once every 3 days, once
every week, once every 2 weeks, once every 3 weeks, or once every 4
weeks. During the period of consecutive days in which Compound
B.sup.2 is administered, Compound B.sup.2 can be administered at
least three times a day, at least twice a day, at least once a day,
or less than once a day, e.g., once every 2 days, once every 3
days, once every week, once every 2 weeks, once every 3 weeks, or
once every 4 weeks.
[0144] Also, contemplated herein is a drug holiday utilized between
the sequential administration of one of Compound A.sup.2 and
Compound B.sup.2 and the other of Compound A.sup.2 and Compound
B.sup.2. As used herein, a drug holiday is a period of days after
the sequential administration of one of Compound A.sup.2 and
Compound B.sup.2 and before the administration of the other of
Compound A.sup.2 and Compound B.sup.2 where neither Compound
A.sup.2 nor Compound B.sup.2 is administered. Suitably the drug
holiday will be a period of days selected from: 1 day, 2 days, 3
days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11
days, 12 days, 13 days and 14 days.
[0145] Sequential administration can also include one of Compound
A.sup.2 and Compound B.sup.2 is administered for two or more
consecutive days and then both of Compound A.sup.2 and Compound
B.sup.2 is subsequently administered for two or more consecutive
days. Sequential administration can include both of Compound
A.sup.2 and Compound B.sup.2 being administered for two or more
consecutive days and then one of Compound A.sup.2 and Compound
B.sup.2 being subsequently administered for two or more consecutive
days
Regarding Sequential Administration:
[0146] Suitably, one of Compound A.sup.2 and Compound B.sup.2 is
administered for from 1 to 30 consecutive days, followed by an
optional drug holiday, followed by administration of the other of
Compound A.sup.2 and Compound B.sup.2 for from 1 to 30 consecutive
days. Suitably, one of Compound A.sup.2 and Compound B.sup.2 is
administered for from 1 to 21 consecutive days, followed by an
optional drug holiday, followed by administration of the other of
Compound A.sup.2 and Compound B.sup.2 for from 1 to 21 consecutive
days. Suitably, one of Compound A.sup.2 and Compound B.sup.2 is
administered for from 1 to 14 consecutive days, followed by a drug
holiday of from 1 to 14 days, followed by administration of the
other of Compound A.sup.2 and Compound B.sup.2 for from 1 to 14
consecutive days. Suitably, one of Compound A.sup.2 and Compound
B.sup.2 is administered for from 1 to 7 consecutive days, followed
by a drug holiday of from 1 to 10 days, followed by administration
of the other of Compound A.sup.2 and Compound B.sup.2 for from 1 to
7 consecutive days.
[0147] Suitably, Compound B.sup.2 will be administered first in the
sequence, followed by an optional drug holiday, followed by
administration of Compound A.sup.2. Suitably, Compound B.sup.2 is
administered for from 3 to 21 consecutive days, followed by an
optional drug holiday, followed by administration of Compound
A.sup.2 for from 3 to 21 consecutive days. Suitably, Compound
B.sup.2 is administered for from 3 to 21 consecutive days, followed
by a drug holiday of from 1 to 14 days, followed by administration
of Compound A.sup.2 for from 3 to 21 consecutive days. Suitably,
Compound B.sup.2 is administered for from 3 to 21 consecutive days,
followed by a drug holiday of from 3 to 14 days, followed by
administration of Compound A.sup.2 for from 3 to 21 consecutive
days. Suitably, Compound B.sup.2 is administered for 21 consecutive
days, followed by an optional drug holiday, followed by
administration of Compound A.sup.2 for 14 consecutive days.
Suitably, Compound B.sup.2 is administered for 14 consecutive days,
followed by a drug holiday of from 1 to 14 days, followed by
administration of Compound A.sup.2 for 14 consecutive days.
Suitably, Compound B.sup.2 is administered for 7 consecutive days,
followed by a drug holiday of from 3 to 10 days, followed by
administration of Compound A.sup.2 for 7 consecutive days.
Suitably, Compound B.sup.2 is administered for 3 consecutive days,
followed by a drug holiday of from 3 to 14 days, followed by
administration of Compound A.sup.2 for 7 consecutive days.
Suitably, Compound B.sup.2 is administered for 3 consecutive days,
followed by a drug holiday of from 3 to 10 days, followed by
administration of Compound A.sup.2 for 3 consecutive days.
[0148] It is understood that a "specified period" administration
and a "sequential" administration can be followed by repeat dosing
or can be followed by an alternate dosing protocol, and a drug
holiday may precede the repeat dosing or alternate dosing
protocol.
[0149] The methods of the present invention may also be employed
with other therapeutic methods of cancer treatment.
[0150] Compound A.sup.2 and Compound B.sup.2 may be administered by
any appropriate route. Suitable routes include oral, rectal, nasal,
topical (including buccal and sublingual), intratumorally, vaginal,
and parenteral (including subcutaneous, intramuscular, intravenous,
intradermal, intrathecal, and epidural). It will be appreciated
that the preferred route may vary with, for example, the condition
of the recipient of the combination and the cancer to be treated.
It will also be appreciated that each of the agents administered
may be administered by the same or different routes and that
Compound A.sup.2 and Compound B.sup.2 may be compounded together in
a pharmaceutical composition/formulation.
[0151] In one embodiment, one or more components of a combination
of the invention are administered intravenously. In one embodiment,
one or more components of a combination of the invention are
administered orally. In another embodiment, one or more components
of a combination of the invention are administered intratumorally.
In another embodiment, one or more components of a combination of
the invention are administered systemically, e.g., intravenously,
and one or more other components of a combination of the invention
are administered intratumorally. In any of the embodiments, e.g.,
in this paragraph, the components of the invention are administered
as one or more pharmaceutical compositions.
[0152] In one aspect methods are provided for the treatment of
cancer, comprising administering to a human in need thereof a
therapeutically effective amount of (i) an anti-ICOS antibody or
the antigen binding portion thereof, in addition to one of more
diluents, vehicles, excipients and/or inactive ingredients, and
(ii) an anti-PD1 antibody or the antigen binding portion thereof or
an anti-PDL1 antibody or the antigen binding portion thereof, in
addition to one of more diluents, vehicles, excipients and/or
inactive ingredients. In one embodiment sequential administration
of an anti-ICOS antibody or the antigen binding portion thereof and
an anti-PD1 antibody or antigen binding portion thereof provides a
synergistic effect compared to administration of either agent as
monotherapy or concurrently. In one embodiment, sequential
administration of an anti-ICOS antibody or the antigen binding
portion thereof and an anti-PDL1 antibody or antigen binding
portion thereof provides a synergistic effect compared to
administration of either agent as monotherapy or concurrently.
[0153] In one embodiment, the anti-ICOS antibody or antigen binding
portion thereof comprises a V.sub.H domain comprising an amino acid
sequence at least 90% identical to the amino acid sequence set
forth in SEQ ID NO:7; and a V.sub.L domain comprising an amino acid
sequence at least 90% identical to the amino acid sequence as set
forth in SEQ ID NO:8.
[0154] In one embodiment, methods of treating cancer are provided
wherein the anti-ICOS antibody or antigen binding portion thereof
is administered at a time interval selected from once every week,
once every two weeks, once every three weeks, and once every four
weeks. In another embodiment, the anti-PD1 antibody or antigen
binding portion thereof or the anti-PDL1 antibody or antigen
binding portion thereof is administered at a time interval selected
from once every week, once every two weeks, once every three weeks,
and once every four weeks. As is understood in the art the start of
administration of either agent can be separated by an interstitial
period. The interstitial period may be 12 hours, one to six days,
one week, two weeks, three weeks, four weeks, five weeks, or six
weeks. By way of example, an anti-ICOS antibody could be
administered on Day 1 of treatment with an interstitial period of
two weeks before the start of anti-PD1 antibody therapy which would
start on Day 14. In one aspect, treatment with said anti-ICOS
antibody could continue with administration of a single IV infusion
at a time interval of, for example, every one, two, three or four
weeks. Similarly, treatment with said anti-PD1 antibody could
continue with administration of a single IV infusion at a time
interval of, for example, every one, two, three or four weeks.
[0155] In one embodiment, the anti-ICOS antibody or antigen binding
portion thereof is administered as an IV infusion. In one
embodiment, the anti-PD1 antibody or antigen binding portion
thereof is administered as an IV infusion. In one embodiment, the
anti-PDL1 antibody or antigen binding portion thereof is
administered as an IV infusion. In one aspect, the anti-ICOS
antibody or antigen binding portion thereof is administered prior
to the anti-PD1 antibody or the antigen binding portion thereof or
the anti-PD1 antibody or the antigen binding portion thereof. In
one embodiment, administration of the anti-PD1 antibody or antigen
binding portion thereof or the anti-PDL1 antibody or antigen
binding portion thereof is initiated at a time point selected from
1 week, 2 weeks, 3 weeks, and 4 weeks after the start of the
administration of said anti-ICOS antibody or antigen binding
portion thereof. In one aspect, the anti-PD1 antibody or antigen
binding portion thereof or the anti-PDL1 antibody or antigen
binding portion thereof is administered prior to the anti-ICOS
antibody or the antigen binding portion thereof. In one embodiment,
the interstitial period between the start of the anti-PD1 antibody
or anti-PDL1 therapy and the start of the anti-ICOS antibody
therapy is selected from 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks,
5 weeks, and 6 weeks.
[0156] In one embodiment, the anti-ICOS antibody or antigen binding
portion thereof and said anti-PD1 antibody or antigen binding
portion thereof or anti-PDL1 antibody or antigen binding portion
thereof are administered to said human until said human shows
disease progression or unacceptable toxicity. In one embodiment,
methods are provided for the treatment of cancer further comprising
administering at least one anti neoplastic agent and/or at least
one immuno-modulatory agent to said human.
[0157] Typically, any anti-neoplastic agent that has activity
versus a susceptible tumor being treated may be co-administered in
the treatment of cancer in the present invention. Examples of such
agents can be found in Cancer Principles and Practice of Oncology
by V. T. Devita, T. S. Lawrence, and S. A. Rosenberg (editors),
10.sup.th edition (Dec. 5, 2014), Lippincott Williams & Wilkins
Publishers. A person of ordinary skill in the art would be able to
discern which combinations of agents would be useful based on the
particular characteristics of the drugs and the cancer involved.
Typical anti-neoplastic agents useful in the present invention
include, but are not limited to, anti-microtubule or anti-mitotic
agents such as diterpenoids and vinca alkaloids; platinum
coordination complexes; alkylating agents such as nitrogen
mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and
triazenes; antibiotic agents such as actinomycins, anthracyclins,
and bleomycins; topoisomerase I inhibitors such as camptothecins;
topoisomerase II inhibitors such as epipodophyllotoxins;
antimetabolites such as purine and pyrimidine analogues and
anti-folate compounds; hormones and hormonal analogues; signal
transduction pathway inhibitors; non-receptor tyrosine kinase
angiogenesis inhibitors; immunotherapeutic agents; proapoptotic
agents; cell cycle signalling inhibitors; proteasome inhibitors;
heat shock protein inhibitors; inhibitors of cancer metabolism; and
cancer gene therapy agents such as genetically modified T
cells.
[0158] Examples of a further active ingredient or ingredients for
use in combination or co-administered with the present methods or
combinations are anti-neoplastic agents. Examples of
anti-neoplastic agents include, but are not limited to,
chemotherapeutic agents; immuno-modulatory agents;
immuno-modulators; and immunostimulatory adjuvants.
EXAMPLES
[0159] The following examples illustrate various non-limiting
aspects of this invention.
Example 1
[0160] The study design of the anti-ICOS antibody/anti-PD1 antibody
concurrent and phased dosing study conducted is shown in FIG. 1.
FIG. 2 is a schematic showing the study procedure 5 of anti-ICOS
antibody/anti-PD1 antibody concurrent and phased dosing study.
Shown at the bottom of FIG. 2 is a table listing antibodies used in
the study. In FIGS. 3-7, FIGS. 8A-8C, FIGS. 9A-9C, and FIGS. 10-14,
"Rt ICOS" refers to "rat anti-ICOS antibody;" "Rt PD1" refers to
"rat anti-PD1 antibody." "Rt IgG2A" refers to "rat IgG2A;" "Rt
IgG2B" refers to "rat IgG2B."
Monotherapy:
[0161] As shown in FIGS. 3, 4, 8B, 10 and 11, rat anti-mouse ICOS
antibody (17G9) 100 .mu.g or 10 .mu.g showed similar tumor growth
rate (FIG. 3, FIG. 4, FIG. 8B) and overall survival (40%) (FIG. 10,
FIG. 11).
[0162] Rat anti-mouse anti-PD1 antibody (200 .mu.g) had no effect
on tumor growth rate (FIG. 3, FIG. 4, FIGS. 8A-8B). Overall
survival was 10% (FIG. 10, FIG. 11).
Combination:
[0163] At day 10, concurrent dosing of anti-ICOS antibody (100
.mu.g or 10 .mu.g) combined with anti-PD1 antibody showed
synergistic anti-tumor efficacy compared to mono or phased dosing
regimen (FIGS. 3-7, 8A-8C, 9A-9C).
[0164] Mice in Group 12 treated with anti-ICOS lead-in/anti-PD1
follow up dosing showed surprising and unexpected increase in long
term survival. Regarding mouse long term survival (day 67 post
1.sup.st dose), 60% of mice from Group 12 (anti-ICOS lead in
followed by 6 doses of anti-PD1) showed complete response (6 mice
were tumor free, 1 mouse found dead due to anti-drug antibodies
(ADA)) (FIG. 10, FIG. 12, FIG. 14). Twenty percent (20%) of mice
from Group 11 (anti-ICOS lead in followed by 6 doses of rat IgG2A)
showed complete response (3 mice were tumor free, 3 mice were found
dead due to ADA) (FIG. 10, FIG. 12, FIG. 14); the data is
comparable to the anti-ICOS monotherapy data. Thirty percent (30%)
of mice from Group 8 (anti-PD1 lead in followed by 3 doses of
anti-PD1+ rat IgG2b) showed complete response (3 mice are tumor
free) (FIG. 10, FIG. 12, FIG. 13); this showed better overall
survival than 3 doses of anti-PD1 (10%, 1 tumor free mouse). Twenty
percent (20%) of mice from Group 9 (anti-PD1 lead in followed by 3
doses of anti-PD1+ anti-ICOS) showed complete response (3 mice were
tumor free, 3 mice were found dead due to ADA) (FIG. 10, FIG. 12,
FIG. 13). ADA occurred at the 4.sup.th and 5.sup.th doses.
[0165] The results described herein in Example 1 were obtained with
the following materials and methods.
Mice, Tumor Challenge and Treatment
[0166] All studies were conducted in accordance with the GSK Policy
on the Care, Welfare and Treatment of Laboratory Animals and were
reviewed by the Institutional Animal Care and Use Committee (IACUC)
either at GSK or by the ethical review process at the institution
where the work was performed. 6-8 week old female BALB/c mice
(Envigo) were utilized for in vivo studies in a fully accredited
AAALAC facility.
[0167] 5.0.times.10.sup.4 cells/mouse of CT26 mouse colon carcinoma
(ATCC CRL-2638) tumor cells were inoculated subcutaneously into the
right flank. Tumor volume and body weight data were collected using
the Study Director.TM. software package (Studylog Systems, South
San Francisco, Calif., USA). Tumor volume was calculated using the
formula: Tumor Volume (mm).sup.3=0.52*1*w.sup.2 where w=width and
l=length, in mm, of the tumor. When tumors reached approximately
50-100 mm.sup.3, mice were randomized into various groups
(n=10/treatment group) based on tumor volume using stratified
sampling method in the Study Log.TM. software prior to initiation
of treatment. Tumor measurement of greater than 2,000 mm.sup.3 for
an individual mouse and/or development of open ulcerations in tumor
and/or body weight loss greater than 20% resulted in mice being
removed from study. Dosing started on randomization day. Mice
received the mouse anti-ICOS (clone 7E.17G9) and/or mouse anti-PD1
(clone RMP1-14) antibodies or saline via intraperitoneal injection
twice weekly starting on randomization day for a total of 3 doses
of anti-ICOS and 3 or 6 doses of anti-PD1 for concurrent and
sequential dosing respectively. In order to evaluate anti-tumor
activity of combining the anti-ICOS and anti-PD-1 monoclonal
antibodies, mice were dosed twice a week with either anti-ICOS
(clone 7E.17G9, rat IgG2b 100 .mu.g) or its isotype control (rat
IgG2b 100 .mu.g) along with anti-PD-1 (clone RMP1-14, rat IgG2a 200
.mu.g) or its isotype control (rat IgG2a 200 .mu.g) concurrently.
For the experiments involving sequential dosing, either dosing with
anti-ICOS antibody started after 3 doses of anti-PD1 which meant
that the the last 3 of the 6 anti-PD1 doses were given in
combination with anti-ICOS or anti-PD1 dosing started after all 3
doses of the anti-ICOS antibody were completed. Appropriate isotype
controls were also employed in a similar dosing regimen.
[0168] Data are plotted using Graphpad.TM. software and Statistical
analysis was performed by Statistician.
Example 2
[0169] Characterization of an IgG4 Anti-ICOS Agonist Antibody that
Elicits T-Cell Activation and Antitumor Responses Alone and with
PD-1 Blockade
[0170] Described in Example 2 is the characterization of the
immune-stimulatory and anti-tumor activity of a humanized
non-depleting anti-ICOS agonist antibody, with an emphasis on the
importance of isotype choice for optimal efficacy and provides
strong rationale for exploring this in cancer patients as a single
agent and in combination with PD-1 checkpoint blockade.
[0171] Inducible T-cell Co-Stimulator (ICOS) is a T-cell-restricted
co-stimulatory receptor whose expression is induced on activated T
cells upon T-cell receptor engagement. We demonstrate that
antibody-mediated ICOS agonism elicits potent T-cell activation,
mobilization of T cells to the tumor site, and antitumor responses
in syngeneic mouse models. Our data indicate that the isotype
choice for the agonist antibody is crucial to avoid Fc-dependent
cytotoxicity and depletion of effector T cells (T.sub.eff), as
observed with an IgG1 version of the antibody tested. Furthermore,
our data suggest that ICOS expression level on regulatory T cells
(T.sub.reg), albeit high, offers a narrow window for selective
depletion of T.sub.regs in most tumors, due to overlapping ICOS
levels on T.sub.eff and the upregulation of ICOS in the presence of
checkpoint blockade. Exploration of isotypes led to the selection
of a humanized IgG4 anti-ICOS agonist antibody (H2L5 IgG4PE) for
clinical development. We present the characterization of the
immunological activity and therapeutic potential of this ICOS
agonist antibody, currently being investigated alone and in
combination with pembrolizumab in a first-in-human clinical
study.
INTRODUCTION
[0172] Inducible T-cell co-stimulator (ICOS) is a co-stimulatory
receptor with structural and functional homology to the
CD28/CTLA-4-Ig superfamily (Hutloff, A. Nature 397:263-266 (1999)).
ICOS expression is upregulated by antigen stimulation and ICOS
signaling induces production of both T.sub.H1 and T.sub.H2
cytokines and effector T-cell (T.sub.eff) proliferation. ICOS
expression has been observed on resting T.sub.H17, T follicular
helper (T.sub.FH) and regulatory T (T.sub.reg) cells; however,
unlike CD28, it is not highly expressed on most resting naive and
memory T-cell populations (Fazilleau, N. et al. Nat Immunol.
8(7):753-61. (2007), Paulos, C. M. et al. Sci Transl Med. 2(55):
55-78. (2010)). ICOS plays a crucial role in the survival and
expansion of T.sub.eff and T.sub.reg during an immune response
(Burmeister, Y. et al. J Immunol. 180(2): 774-82. (2008)) and has
been shown to be critical for the development and function of
T.sub.H17 (Paulos, C. M. et al. Sci Transl Med. 2(55): 55-78.
(2010), Guedan, S. et al. Blood 124(7): 1070-80. (2014)).
[0173] Emerging data from patients treated with anti-CTLA-4
antibodies suggest ICOS-expressing memory T cells may help mediate
antitumor immune responses and long-term survival (Liakou, C. I. et
al. Proc Natl Acad Sci USA. 105(39): 14987-92. (2008); Di Giacomo,
A. M. et al. Cancer Immunol Immunother. 62(6): 1021-8. (2013);
Carthon, B. C. et al. Clin Cancer Res. 16(10): 2861-71. (2010);
Vonderheide, R. H. et al. Clin Cancer Res. 16(13): 3485-94.
(2010)). ICOS has been shown to be critical for anti-CTLA-4
antitumor activity in mice (Fu, T. He, Q., Sharma, P. Cancer Res.
71(16): 5445-54. (2011); Fan, X, et al. J Exp Med. 211(4):715-25.
(2014)) and prior reports support the concept that activating ICOS
on CD4 and CD8 T cells using recombinant murine ICOS ligand has
antitumor potential (Ara, G. et al. Int. J Cancer. 103(4): 501-7
(2003)). Human ICOS ligand (ICOS-L) has been shown to bind both
CTLA-4 and CD28 in addition to ICOS, which limits the potential use
of recombinant ICOS-L as a therapeutic in humans (Yao, S. et al.
Immunity 34(5), 729-40. (2011)); necessitating an alternative
therapeutic approach to activate ICOS in patients with cancer.
[0174] Here, we describe the immunologic and antitumor
characterization of a first-in-class humanized IgG4 anti-ICOS
agonist monoclonal antibody (mAb) H2L5 IgG4PE, designed to deliver
optimal ICOS agonism via Fc gamma receptor (Fc.gamma.R)
cross-linking, with minimal antibody-dependent cellular
cytotoxicity (ADCC) and phagocytosis activity; thereby reducing the
risk of T.sub.eff depletion. The comprehensive preclinical data
described herein, support clinical testing of H2L5 IgG4PE,
currently being investigated alone and in combination with
pembrolizumab in a first-in-human clinical study.
Results
[0175] Development of a Potent and Selective Anti-Human ICOS
Agonist Monoclonal Antibody (mAb)
[0176] We undertook the generation of an agonistic anti-human ICOS
mAb by immunizing mice with ICOS extracellular domain. One of these
mAb was humanized and expressed as a human IgG4 with 2 Fc mutations
(glutamic acid for leucine at residue 235) (Kabat, E. A., et al.
Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept.
of Health and Human Services, Bethesda, Md., NIH Publication no.
91-3242. (1991)) and substitution of proline for serine at residue
228 (EU numbering) to reduce antigen binding fragment (Fab) arm
exchange with native IgG4 (Manjula, P. et al. The Journal of
Immunology. 164:1925-1933. (2000), Rispens, T. et al. J. Am. Chem.
Soc. 133 (26):10302-10311. (2011)). The resulting H2L5 IgG4PE
hereafter referred to as "H2L5".
[0177] H2L5 bound to human ICOS with an affinity of 1.34 nM (FIG.
15A), which is approximately 17-fold higher than the native
ICOS-L/CD275 interaction (FIG. 15B). H2L5 did not bind to murine
ICOS or to human CD28 or CTLA-4, the two nearest structurally
related protein. This contrasts with the native human ICOS-L, which
binds both CTLA-4 and CD28 (Yao, S. et al. Immunity 34(5), 729-40.
(2011)). H2L5 blocked ICOS/ICOS-L binding by flow cytometry and
competed partially (.ltoreq.50%) with ICOS-L in binding to ICOS at
concentrations above 1 g/mL in MSD immunoassays (FIGS. 22A-22B).
H2L5 also bound to both CD4 and CD8 T-cells in activated PBMC
samples from healthy human donors (FIG. 15C). ICOS has previously
been shown to activate AKT in response to ICOS-L binding in human T
cells (Okamoto, N. et al. Biochem Biophys Res Commun. 310(3):
691-702. (2003)); pre-activated primary human CD4 T cells
demonstrated an increase in pAKT and pGSK30 in response to
treatment with H2L5 (FIG. 15D). H2L5 significantly increased CD4
and CD8 T-cell activation, when used in a plate-bound format
together with anti-CD3, as measured by CD69 expression (FIG. 15E)
and proliferation (FIG. 15F). Minimal activation was observed with
H2L5 in the absence of anti-CD3 treatment, indicating that it does
not have superagonist activity under these assay conditions.
[0178] The plate-bound H2L5 antibody induced a dose-dependent
increase in T.sub.H1, T.sub.H2 and T.sub.H17 cytokines,
IFN-.gamma., TNF-.alpha., IL-17a, IL-10, IL-6 and to a lesser
extent IL-2, IL-5 and IL-13 in PBMC from healthy donors (HD) (FIG.
15G, FIG. 23A, FIG. 33). A similar profile of cytokine induction
was observed in PBMC from NSCLC patients, with strong induction of
IFN-.gamma., and lower levels of other cytokines including
TNF-.alpha., IL-10 and IL-2 (FIG. 15H, FIG. 34). A dose-dependent
increase in T-cell activation markers: CD25, OX40 and CD69 on both
CD4 and CD8 T cells was also observed with HD following stimulation
with plate-bound anti-CD3 and H2L5 (FIG. 23B). With isolated human
CD3.sup.+ T cells, treatment with H2L5 led to a significant
increase in the mRNA expression of the T.sub.H1 transcription
factor T-Bet (FIG. 15I) as well as the cytotoxic molecule
Granzyme-B (FIG. 15J). A significant decrease in L-Selectin
expression was observed, indicating a transition towards an
activated effector phenotype (FIG. 15K). The ability of the
plate-bound H2L5 antibody to costimulate T cells isolated ex vivo
from disaggregated tumors in the presence of anti-CD3 after 6 days
of culture, was also assessed. A concentration dependent and robust
increase in IFN-.gamma. was seen in 9/10 donors (FIG. 15L), along
with less robust induction of IL-17 and IL-10 compared with healthy
PBMC, as well as low-undetectable levels of T.sub.H2 cytokines
(IL-5 and IL-13) (FIG. 24A-D). Significant increases in activation
markers OX40 (FIG. 15M), CD25 (FIG. 15N) and LAG3 (FIG. 25A) were
observed on CD8 T cells, in addition to a modest increase in
CD8+PD-1+ cells and CD4+CD25+Foxp3+(T.sub.reg) cells (FIGS.
25B-25D). Only low percentage of ICOS-L expressing cells were
observed with most donors (FIG. 25C)
[0179] Altogether, these data show that H2L5 is a potent ICOS
agonist, capable of driving T-cell activation and proliferation,
but is not a superagonist capable of driving T-cell activation in
the absence of TCR stimulation.
Antibody Isotype and Fc.gamma.R-Engagement is Critical for H2L5
Function
[0180] Fc.gamma.R-mediated crosslinking is critical for agonist
antibody function (Dahal, L. N. et al. Immunol Rev. 268(1): 104-22.
(2015); Furness A. J. et al. Trends in Immunology 35(7): 290-298
(2014)). The results described in FIG. 15 utilized plate-bound
antibody, which overcomes the need for Fc.gamma.R cross-linking and
suggests that an antibody isotype capable of engaging Fc.gamma.R
and mediating crosslinking is key to achieving optimal ICOS
agonism. To formally assess this, we cloned the heavy and light
chain variable regions of H2L5 and expressed them as different
human IgG isotypes (IgG1, IgG2, IgG4PE and IgG1 Fc-disabled [amino
acid (AA) substitutions L235A and G237A) (Bartholomew, M. et al.
Immunology 85(1): 41-8 (1995)). The binding of the different H2L5
isotype variants was determined against human Fc.gamma.RI,
Fc.gamma.RIIa (H131), Fc.gamma.RIIa (R131), Fc.gamma.RIIb,
Fc.gamma.RIIIa (V158) and Fc.gamma.RIIIa (F158) and demonstrated
expected patterns of binding (FIG. 35). The IgG4PE contained two AA
substitutions from native human IgG4; glutamic acid for leucine at
residue 235 (Kabat, E. A., et al. Sequences of Proteins of
Immunological Interest, 5th Ed. U.S. Dept. of Health and Human
Services, Bethesda, Md., NIH Publication no. 91-3242. (1991)) and
substitution of proline for serine at residue 228 (EU numbering) to
reduce antigen binding fragment (Fab) arm exchange with native IgG4
(Manjula, P. et al. The Journal of Immunology. 164:1925-1933.
(2000), Rispens, T. et al. J. Am. Chem. Soc. 133 (26):10302-10311.
(2011)) and decrease binding to activating Fc.gamma.R and C1q,
while retaining binding to the inhibitory Fc.gamma.RIIb. In PBMC
assays the H2L5 IgG1 antibody decreased both CD4 and CD8 T-cell
proliferation when added in solution in greater than 50% of donors
tested (FIG. 16A). In contrast, IgG2, IgG4PE and Fc-disabled
isotype variants of H2L5 did not result in substantial inhibition
of either CD4 or CD8 T-cell proliferation in any donors tested,
while the H2L5 IgG4PE format resulted in increased proliferation in
a subset of the donors (FIG. 16A). We next tested whether the
inhibitory effect of H2L5 IgG1 was due to ADCC via NK cells in the
PBMC mixture. In PBMCs from 10 healthy donors (HD), the inhibitory
effect of H2L5 IgG1 on both CD4 and CD8 populations was lost when
NK cells were removed from the PBMC pool (FIG. 16B). The H2L5
isotype variants were also tested in a reporter assay that detects
engagement of Fc.gamma.RIIIa, the primary activating Fc.gamma.R
responsible for NK-mediated ADCC in humans. While the H2L5 IgG1
induced a significant increase in luciferase signaling, when
incubated with activated T cells, neither the H2L5 IgG4PE nor H2L5
Fc-disabled antibodies induced Fc.gamma.RIIIa-mediated signaling
(FIG. 26A). Additionally, H2L5 IgG1 induced T-cell death in an
NK-dependent manner whilst neither IgG4PE nor Fc-disabled H2L5
resulted in any significant increase in cell death as compared to
isotype controls (FIG. 16C).
[0181] Previous studies have reported that receptor density may
influence the susceptibility of T cells to killing by ADCC, leading
to potential preferential depletion of different T-cell subsets,
which may differ in the tumor microenvironment compared to the
lymphoid tissue (Furness A. J. et al. Trends in Immunology 35(7):
290-298 (2014)). The level of expression of ICOS on CD4, CD8 and
T.sub.reg freshly isolated from different tumors was determined by
flow cytometry. Although there was higher expression observed on
T.sub.reg vs CD4 and CD8 T cells, this was heterogeneous with some
tumors showing overlapping levels between these populations;
consequently, ICOS high expression was not a distinct feature of
T.sub.reg (FIG. 16D). To further evaluate the relative contribution
of Fc isotype on potential depletion by ADCC, the CD4, CD8 and
T.sub.reg cells were purified directly from different cancer
patients and the level of ICOS receptor density was correlated with
ability of the IgG1 or IgG4PE isotype of H2L5 to stimulate
Fc.gamma.RIIIA in an ex vivo reporter assay (FIG. 16E, FIG. 26B). T
cells isolated from tumors did not stimulate Fc.gamma.RIIIa when
incubated with the H2L5 IgG4PE isotype; whereas incubation with
H2L5 IgG1 did lead to some (variable) stimulation. In some tumors,
FcR.gamma.IIIa receptor engagement was seen with T.sub.reg, CD4 and
CD8, especially at doses of 1-10 .mu.g/mL supporting the idea that
selective ADCC deletion of T.sub.reg without affecting CD4 and CD8
may not be universally possible in all tumors (e.g. Breast 1001202
patient sample conventional CD4 T cells induced similar stimulation
to the T.sub.reg at doses of 1-10 .mu.g/ml; FIG. 16F, FIG.
26B).
[0182] Based on the above data, the isotype selected for
development was the engineered IgG4PE antibody, H2L5.
H2L5 Induces Fc.gamma.R-Mediated Agonism of TCR Dependent T-Cell
Activation.
[0183] H2L5 was tested with isolated human CD4 T cells in both a
plate-bound (immobilized antibody) format as well as in solution.
H2L5 in the immobilized format, which simulates membrane-bound
Fc.gamma.R-dependent crosslinking, induced significantly greater
levels of IFN-.gamma. compared with the soluble antibody (FIG.
17A). The importance of Fc.gamma.R engagement for optimal H2L5
agonist activity was further confirmed in an activated human PBMC
assay, where H2L5 resulted in >2-fold induction of IFN-.gamma.;
whereas the Fc-disabled version of H2L5 had no cytokine induction
activity compared with the isotype control (FIG. 17B). The IgG4PE
and Fc-disabled versions of H2L5 were also tested in a modified
mixed lymphocyte reactions (MLR). The H2L5 IgG4PE mAb provided
>2-fold induction of IFN-.gamma. whereas the Fc-disabled H2L5
mAb had no activity compared with the isotype control (FIG. 17C).
Next, a CD4 T cell/CD14 monocyte donor-matched co-culture assay was
utilized to determine whether Fc.gamma.R-expressing monocytes
increased the agonist potential of soluble H2L5. Like the MLR assay
format, H2L5 only induced IFN-.gamma. when tested as an IgG4PE
isotype; the Fc-disabled antibody showed no significant cytokine
induction compared with the isotype control. The addition of
monocytes, which are known to express Fc.gamma.Rs including
Fc.gamma.RII isoforms, resulted in a significant increase in H2L5
IgG4PE-induced cytokine production compared with T cells alone.
Interaction with Fc.gamma.RIIB has been shown to be critical for
the agonistic activity of other immunomodulatory antibodies
targeting TNF-.alpha. family receptors as well as CD28
(Bartholomew, M. et al. Immunology 85(1): 41-8 (1995);
Bartholomaeus, P. et al. J Immunol. 192(5): 2091-8. (2014)).
Conversely, the addition of an Fc.gamma.R-blocking antibody
completely inhibited the H2L5-induced cytokine induction (FIG.
17D). These results indicate that H2L5 can achieve Fc.gamma.R
engagement likely via the Fc.gamma.RIIB as seen with other IgG4
agonist antibodies (Bartholomaeus, P. et al. J Immunol. 192(5):
2091-8. (2014), Hussain, K. et al. Blood 125 (1): 102-110 (2015)),
while avoiding ADCC killing of ICOS+ T cells, as seen with the IgG1
isotype.
[0184] To assess its localization and mobilization at the cell
surface, H2L5 was fluorescently labeled, added to primary activated
human CD3+ T cell cultures, alongside DCs and imaged. Following
binding, H2L5 rapidly polarized on the T cell surface. The
mobilized T cells began scanning the culture until binding with a
dendritic cell (DC) was initiated. In instances where T cells were
in cellular contact with DCs, H2L5 accumulated at the point of
contact (FIG. 17E). Additional studies using co-cultures of human
DC and T-cells demonstrated that H2L5 was rapidly co-localized with
CD28 and to a lesser extent CD4 at the polarized caps of activated
T cells as well as the subsequent immune synapses that formed upon
T-cell binding to DC (FIG. 17F). These results indicate that ICOS
induces human T-cell mobilization and is co-located at the immune
synapse following T cell activation.
H2L5 Induces an Effector Memory Phenotype and Antitumor Activity In
Vivo
[0185] The in vivo functionality of H2L5 was evaluated in a human
PBMC engrafted NSG mouse model implanted with A2058 tumors. This
model induces a Graft-versus-Host Disease (GVHD) response and has
been used previously to study effector and memory T-cell activity
(23). In the blood of H2L5-treated mice, the number of human T
cells decreased in a dose-dependent manner (FIG. 18A), while a
corresponding increase in CD69 expression (representing T-cell
activation) was observed (FIG. 18B). The Fc-disabled version of
H2L5 showed similar, albeit weaker, trends than H2L5 IgG4PE
suggesting that the disappearance of cells was not due to ADCC.
H2L5 induced a dose-dependent increase in CD4+CD45R0+CD62L-effector
memory (TEM) cells (FIG. 18C), and CD8.sup.+CD45RO.sup.-CD62L.sup.-
terminally differentiated CD8 effector cells (TEMRA) (FIG. 18D).
H2L5 was next tested in human PBMC engrafted NSG mice harboring
either HCT116 or A549 tumors. Detection of H2L5 binding to ICOS+ T
cells (CD4, CD8 and T.sub.reg), by a human anti-IgG4 fluorescent
labelled antibody, was observed in blood and tumor at doses of 0.4
and to lesser extent 0.04 mg/kg demonstrating target engagement in
mice bearing the A549 tumors (FIG. 18E, FIGS. 27A-27B). Mice
treated with anti-PD-1 IgG4 antibody (Keytruda) also showed the
detection of bound antibody using the same detection reagent.
Treatment of mice with H2L5 was associated with an increase in the
CD8:T.sub.reg ratio in the A549 tumors, comparable to that seen in
mice treated with anti-PD-1. (FIG. 18F). H2L5 resulted in
significant tumor growth inhibition in both HCT116 and A549 tumor
models (FIGS. 18G-18H). In the A549 model where the GVHD response
was less severe, tumor growth inhibition resulted in dose-dependent
increase in survival beyond 50 days (FIG. 181). These experiments
suggest that doses of 0.4 mg/kg, which correlate with successful
engagement of the ICOS receptor, result in subsequent
pharmacological effects associated with T-cell activation in blood
and tumor and reduction of tumor growth.
The Fc Isotype of Murine Anti-ICOS Antibody Influences Efficacy in
Syngeneic Tumors
[0186] Studies in the literature using CTLA-4, PD-L1, OX40 and CD40
have shown that selection of the Fc isotype of mAbs can
significantly influence efficacy in different tumor models (Dahal,
L. N. et al. Immunol Rev. 268(1): 104-22. (2015); Furness A. J. et
al. Trends in Immunology 35(7): 290-298 (2014)). To generate a
surrogate mouse anti-ICOS antibody equivalent to a human IgG4 in
terms of Fc.gamma.R binding, (FIG. 36) the anti-mouse ICOS antibody
7E-17G9 was cloned into murine (m) IgG1 and mIgG2a isotypes and
tested in 2 different tumor models. The 7E-17G9 antibody showed
agonistic activity in plate-bound format with anti-CD3 (FIG. 28).
In the EMT6 model the mIgG1 antibody showed greater efficacy than
the mIgG2a especially at higher doses (>5 mg/kg, 100
.mu.g/mouse) with both survival (FIG. 19A) and tumor growth
inhibition (FIG. 29A). However, both isotypes showed only modest
dose-dependent efficacy as monotherapy in the CT26 model (FIG. 19B,
FIG. 29B). As described above for the human IgG1, the mIgG2a
depleting antibody may be less effective than the mIgG1, since it
has the potential to deplete both T.sub.eff and T.sub.reg. A
significantly higher CD8:T.sub.reg ratio (FIG. 19C) was observed
for EMT6 vs CT26, prior to treatment (100 mm.sup.3) and both EMT6
and CT26 models showed an increase in the percentage of ICOS
positive CD4 and CD8 and T.sub.reg cells in tumor vs spleen (FIG.
19D, FIG. 30) but higher percentage of ICOS CD8 positive cells were
observed in spleen in EMT6 vs CT26 (80% vs 10%). Although high
levels of ICOS expression on T.sub.reg from tumor-infiltrating
lymphocytes (TILs) were observed for both EMT6 and CT26 tumors,
ICOS levels on CD8 TILs were approximately 10-fold higher in EMT6
than CT26 (30,000 vs 3000 MFI). This suggests that high ICOS
expression on CD8 in both periphery and tumor may be associated
with response with the agonistic activity of the mIgG1 antibody in
the EMT6 model (FIGS. 19E-19G). To further explore mechanisms of
the agonist anti-ICOS mAb in mice bearing EMT6 breast tumors,
effects on TCR diversity were investigated; significant changes in
the number of unique circulating TCR clones and a corresponding
increase in TCR clonality in the blood of ICOS mAb treated mice was
noted (FIGS. 31A-31C). Most clones that expanded in mouse blood in
response to ICOS agonist mAb treatment were also found in tumors
(FIG. 19H). These findings indicate that a small pool of
tumor-reactive T-cell clones expand in response to ICOS mAb
treatment.
Characterization of the ICOS/ICOS-L Pathway in Human Cancers
[0187] To further explore the translation of an ICOS agonist mAb as
an anti-tumor therapeutic antibody, human solid tumors from the
TCGA database were ranked by ICOS mRNA expression (FIG. 26).
Highest expression was observed in head and neck, gastric,
esophageal, melanoma, NSCLC, cervical and breast cancer. Expression
was confirmed in NSCLC by singleplex IHC (FIG. 32). As the H2L5
agonist mAb mode of action is designed to phenocopy ICOS-L
activity, the co-expression of mRNA for ICOS and ICOS-L was
analyzed in these tumor types (FIG. 20A). ICOS expression was often
not co-expressed with ICOS-L, supporting the hypothesis that H2L5
may augment the low level ICOS signaling in these tumors. We also
assessed the relative expression of PD-L1 in the same samples.
Expression of PD-L1 has been associated with increased T-cell
infiltration and used as a predictive biomarker to enrich for
patients responding to anti-PD-1/PD-L1 treatment in different
indications. Overall there was a clear association between PD-L1
and ICOS expression but this was variable between different
indications (FIG. 20A). These results were confirmed by IHC
staining for expression of CD4, CD8 and FOXP3 and tended to
localize with ICOS in immune infiltrates in NSCLC (FIG. 20B).
[0188] The presence of key cell types in the tumor microenvironment
was analyzed by flow cytometry in biopsies from different tumor
types. Of the CD45+ leukocyte population, CD3 T cells appeared to
be the dominant cell type, ranging from 20-80%; other cell types
such as B cells, macrophages, monocytes, NK cells and DC were also
present (FIG. 20C). These cells types express Fc.gamma.R including
Fc.gamma.RIIb, which may provide the cross-linking required for
agonistic activity of H2L5 in the tumor microenvironment (Furness
A. J. et al. Trends in Immunology 35(7): 290-298 (2014)). The
composition of the T-cell sub-populations averaged CD4 (68%), CD8
(30%) and T.sub.reg (2%) although there was considerable
heterogeneity between different tumors. When tumor types were
analyzed separately, the heterogeneity between CD8 and T.sub.reg
was clear with NSCLC and RCC showing a high CD8/T.sub.reg ratio
(FIG. 20D). Further analysis by multiplex IHC was performed to
characterize ICOS expression of different T-cell sub-types.
Co-expression of ICOS was observed on a proportion of CD3+PD-1+
cells, especially in head and neck, esophageal, NSCLC and melanoma
supporting a rationale for combination treatment with anti-PD-1
therapies (FIGS. 20E-20F).
[0189] Next, the effects of H2L5 costimulation on gene expression
by purified human T cells was determined using the Human
PanCancer-Immune profiling panel to identify an ICOS gene
signature. Compared with anti-CD3 alone, 120 genes were
differentially induced with 85 upregulated and 35 down-regulated
(FIG. 20G). Several immune related genes or pathways were induced
by H2L5 compared to anti-CD3 alone including T.sub.H1 cytokines,
and chemokines, T-cell function and cytotoxicity, and TNF family
members (FIG. 38). The top genes identified from EMT6 mouse tumors
treated with 7E.17G9 that overlapped with the human ICOS-induced
signature are shown in FIG. 20H. This information is being used to
guide development of an ICOS transcriptional signature to monitor
pharmacodynamic effects of H2L5 in early clinical studies.
ICOS Agonist Treatment Induces PD-1/PD-L1 in Tumors and
Demonstrates Increased Activity in Combination with Anti-PD-1
Blockade
[0190] PD-L1, a known IFN-.gamma. responsive gene, as well as PD-1,
increased significantly in the tumors of ICOS mAb treated mice
(FIGS. 21A-21B). Human PBMCs were collected from six cancer
patients and treated with H2L5, which resulted in a significant
increase in PD-1 expression on both CD4 and CD8 T cells (FIG. 21C).
In addition, NSCLC and melanoma patients treated with anti-PD-1
therapies showed an increase in ICOS expression on CD4 T cells in
peripheral blood compared with pre-treatment (FIG. 21D). Therefore,
we tested whether combination with a PD-1 blocking antibody could
augment the antitumor activity of the ICOS agonist mAb. The ICOS
agonist mAb (7E17G9 mIgG1 isotype) was dosed alone or in
combination with anti-PD-1 antibody in mice with established EMT6
tumors (150 mm.sup.3). Combination resulted in an increased
antitumor response and long-term survival (90% of mice) as compared
with monotherapy treatment with ICOS or PD-1 antibodies alone (FIG.
21E). The combination of H2L5 and anti-PD-1 (pembrolizumab) was
also assessed in the humanized mouse model and resulted in enhanced
antitumor response to A549 tumors compared with monotherapy alone
(FIG. 21F) These data show that the addition of an ICOS agonist
antibody significantly improved the antitumor activity induced by a
PD-1 antibody.
[0191] H2L5 was further tested alone or in combination with
pembrolizumab in primary resected tumors from 6 patients with NSCLC
in an ex vivo assay. While treatment with H2L5 alone resulted in a
significant increase in IFN-.gamma. in 4/6 of the NSCLC tumor
samples tested, the combination of H2L5 and pembrolizumab resulted
in a significant increase in IFN-.gamma. as compared to
pembrolizumab alone and an increase in 5/6 samples as compared to
H2L5 treatment alone (FIG. 21G). The H2L5 combination with
pembrolizumab was also tested in a modified allogeneic human MLR
assay where combination treatment resulted in increased IFN-.gamma.
levels as compared to either agent alone in 3/3 different healthy
donor pairs (FIG. 21H).
DISCUSSION
[0192] We have presented the first full characterization of the
immunological activity and therapeutic potential of a
first-in-class, humanized IgG4 anti-ICOS agonist mAb, H2L5. We have
demonstrated that the H2L5 IgG4PE agonist antibody induces
significant activation and clonal expansion of both CD4 and CD8 T
cells in vitro and in vivo. These T cells have increased effector
function through increased expression of T.sub.H1 cytokines such as
IFN-.gamma., as well as increased production of cytotoxic factors
such as Granzyme B. ICOS antibody-activated T cells displayed
increased tissue-homing to tumors with significant accumulation and
infiltration resulting in antitumor responses. Prior reports using
ICOS.sup.-/- and ICOS-L.sup.-/- mice as well as blocking antibodies
to ICOS-L have demonstrated the importance of ICOS for the
expansion, survival and function of both CD4 and CD8 TEM cells in
mice (4, 24-25). Additionally, patients with common variable immune
deficiency, which is characterized by a homozygous loss of ICOS,
have been found to have fewer memory T cells, specifically those
which are CD62L.sup.low (26). Our studies using a novel human
ICOS-specific agonist antibody have confirmed the role of ICOS for
inducing this population of memory T cells, providing a viable
therapeutic approach for targeting this important mechanism in
humans.
[0193] We show that the engineered form of IgG4 that incorporates
the mutations S228P and L235E (EU numbering) relative to the native
human IgG4 is the preferred antibody isotype over IgG1 for
achieving agonist function against human ICOS. These AA changes
prevent heterogeneous exchange with native IgG4 (Rispens, T. et al.
J. Am. Chem. Soc. 133 (26): 10302-10311. (2011)). The IgG4PE
isotype also has reduced binding to activating Fc.gamma.R and C1q
compared to human IgG1, thereby diminishing the cytotoxic potential
of H2L5 that could result in depletion of ICOS-positive T cells
through antibody-dependent or complement-dependent mechanisms,
respectively (Manjula, P. et al. The Journal of Immunology.
164:1925-1933. (2000)). Our in vitro studies have shown that the
IgG1 isotype of H2L5 (the initial isotype of H2L5 planned for
development) is able to kill activated CD4 and CD8 T cells
expressing high levels of ICOS, as well as reduce their
proliferation in an NK-dependent manner; this was not seen not seen
with the IgG4PE isotype. Importantly, the IgG4PE isotype retains
functional binding to Fc.gamma.RIIb (the inhibitory Fc.gamma.R),
critical for enabling agonist activity against several stimulatory
immune receptors (Bartholomaeus, P. et al. J Immunol. 192(5):
2091-8. (2014); Hussain, K. et al. Blood 125 (1): 102-110 (2015),
Aalberse, R. C and Schuurman, J. Immunology 105(1): 9-19. (2002);
Schuurman J. and Parren P. W. Curr Opin Immunol. (2016); White A.
L. et al. J Immunol. 187(4):1754-63. (2011); White A. L. et al. J
Immunol 193 (4): 1828-1835 (2014); Dahal R. et al. Cancer Cell.
29(6):820-31. (2016); Yu X. et al. Cancer Cell 33 (4): 664-675
(2018)), which may also be essential for ICOS agonist activity and
associated antitumor effects in humans. The selection of the IgG4PE
isotype was further supported by in vivo studies using the
anti-murine ICOS 7E17G9 surrogate antibody, where the murine IgG1
isotype showed greater efficacy than the deleting IgG2a antibody in
the EMT6 syngeneic model. Murine IgG1 has a similar profile to
human IgG4, with low binding to activating Fc.gamma.R receptors,
yet retaining binding to some Fc.gamma.-receptors including,
inhibitory Fc.gamma.RIIB and inducing Fc-dependent crosslinking to
improve agonism of the anti-ICOS antibody; whereas the murine IgG2a
can bind the activating Fc.gamma.R, like human IgG1, and is able to
mediate effective deletion. Studies performed with CTLA-4, PD-L1,
OX40 and CD40 have shown that selection of the Fc isotype of mAbs
can significantly influence efficacy in different tumor models;
however, this needs to be optimised for each target, depending on
relative expression levels on different cell types (e.g. CD8 vs
T.sub.eff vs T.sub.reg) and mode of action of the antibody
(agonism/deletion) and epitope specificity (Dahal, L. N. et al.
Immunol Rev. 268(1): 104-22. (2015), Furness A. J. et al., Trends
in Immunology 35(7): 290-298 (2014), Yu X. et al. Cancer Cell 33
(4): 664-675 (2018)). Ex vivo human tumors contain varying
proportions of B cells, macrophages and DCs, known to express
Fc.gamma.RIIB, which is critical to mediate the Fc.gamma.R
crosslinking required for H2L5 in the tumor microenvironment
(Furness A. J. et al. Trends in Immunology 35(7): 290-298 (2014),
Hussain, K. et al. Blood 125 (1): 102-110 (2015), Aalberse, R. C
and Schuurman, J. Immunology 105(1): 9-19. (2002); Schuurman J. and
Parren P. W. Curr Opin Immunol. (2016); White A. L. et al. J
Immunol. 187(4):1754-63. (2011); White A. L. et al. J Immunol 193
(4): 1828-1835 (2014); Dahal R. et al. Cancer Cell. 29(6):820-31.
(2016); Yu X. et al. Cancer Cell 33 (4): 664-675 (2018)). A balance
in favour of inhibitory Fc.gamma.RIIB vs activating Fc.gamma.R is
often seen in the immunosuppressive environment of human tumors,
which may favor the cross-linking of H2L5 and enhance its agonist
activity (Furness A. J. et al. Trends in Immunology 35(7): 290-298
(2014), Dahal, L. et al. Cancer Research 77 (13) 3619-3631
(2017)).
[0194] Given the agonist activity of H2L5 described above, one
factor which must be considered, is the expression of ICOS on
T.sub.reg cells in the tumor microenvironment. The relationship of
ICOS positive T-cell subsets on response to the murine 7E. 17E7
IgG1 surrogate antibody in the EMT-6 and CT26 murine tumor models
was explored. A higher ratio of ICOS+CD8:T.sub.reg was observed at
baseline in the EMT6 model vs CT26 in tumors, which may be one
factor leading to greater efficacy in EMT6 model observed with
7E.17G9 IgG1. Similarly, in the humanized mouse model tumor
reduction by H2L5 was associated with an increased CD8:T.sub.reg
ratio. In this model, the response to treatment with H2L5
monotherapy was similar to anti-PD-1 treatment. These results
suggest that the presence of ICOS-positive T.sub.reg does not
preclude the ability of an ICOS agonist to provide therapeutic
benefit.
[0195] Human tumors express varying proportions of CD4 and CD8
T.sub.eff and T.sub.reg, with considerable variability between
tumor types. The percentage of ICOS positive cells and the level of
ICOS expression was found to be heterogeneous between different
cell types, with a trend for higher ICOS levels on T.sub.reg
although in many patients there was overlap between expression of
ICOS on T.sub.reg vs CD4 and CD8 T cells. The IgG1 isotype but not
the IgG4 isotype of H2L5 anti-ICOS antibody was able to bind and
induce activation of the Fc.gamma.RIIIA luciferase reporter assay
with of ex vivo purified T.sub.reg, and to some extent CD8 and CD4
cells. While the IgG1 mediated activity in this assay system was
found to correlate with ICOS receptor density, as has been reported
with other targets such as CTLA-4, OX40 and GITR (19), the
differential expression of ICOS on T.sub.reg vs T.sub.eff was less
prominent. Furthermore, since ICOS expression on T.sub.eff is
enhanced with anti-PD-1 and anti-CTLA-4 treatment, this suggests
that there may not be a large therapeutic window for the IgG1
isotypes to mediate selective depletion of T.sub.reg over T.sub.eff
in vivo. Based on the data described above, strategies for the
development of H2L5 include selection of tumor types which have a
high CD8:T.sub.reg ratio and high ICOS expression on CD8 T cells,
(e.g., NSCLC) and development of rational combinations with agents
that decrease the abundance of, or limit the function of,
T.sub.reg.
[0196] A rational combination partner supported by our data is a
PD-1/PD-L1 blocking antibody. ICOS agonist antibody treatment
significantly induced PD-1 on human T cells as well as PD-1 and
PD-L1 expression in tumors of treated mice; furthermore, anti-PD-1
treatment was also shown to induce expression of ICOS on CD4 and
CD8 T.sub.eff cells. Like the combinatorial activity observed in
mice, the human ICOS agonist H2L5 in combination with the PD-1
blocking antibody, pembrolizumab, demonstrated increased cytokine
production relative to either agent alone in ex vivo human immune
cell assays. This robust induction of IFN-.gamma. by H2L5 IgG4PE
supports the rationale of anti-PD-1 combination as IFN-.gamma. is
known to act on negative feedback by up-regulation of PD-L1
(Mandal, M. et al. Clinical cancer Research 22(10):2329-2334).
[0197] Single-agent treatment with anti-PD-1 or anti-PD-L1
antibodies has demonstrated response rates between 15-30% across
many solid tumors (e.g. bladder, head and neck, lung) (Hoos, A.
Nat. Rev. Drug Disc. 15(4):235-47. (2016)). Emerging clinical data
using PD-1 or PD-L1 antibodies in combination with other agents has
shown signals of increased activity in some settings, however with
substantial added toxicity in some instances (Larkin, J. et al. N
Engl J Med. 373:23-34. (2015); Forde P. M., et al. New England
Journal of Medicine (2018); Xu, X. et al. Int. J Cancer. 142:
2344-2354 (2018)). Several predictive biomarkers have shown
mechanism of response and resistance to anti-PD-1 treatment and
support rationale for combinations (Gibney G. T. et al. Lancet
Oncology 17(12): 542-551(2016); Tumeh P. C. et al. Nature. 515
(7528): 568-71. (2014); Taube, T. M. et al. Clin. Cancer Research.
20 (19):5064-5074 (2014)). It has been shown the tumor mutational
burden, is a correlate of the generation of neoantigens which
stimulate expansion of the endogenous tumor specific repertoire and
is associated with response to anti-PD-1 therapy (Schumacher, T. N.
and Schreiber, R. D. Science 348: 69-74 (2015)). The clonality and
degree of T-cell infiltration in tumors has recently been shown to
be an important positive predictor for immunotherapy outcomes in
cancer (Xu, X. et al. Int. J Cancer. 142: 2344-2354 (2018)). Our
findings demonstrate that with the murine surrogate antibody, TCR
clones were expanded and shared between both blood and tumor.
Furthermore, H2L5, which induces T-cell proliferation, expansion,
and tumor infiltration, may be complimentary to other immunotherapy
agents with distinct mechanisms of action.
[0198] Results described in Example 2 were obtained using the
following materials and methods:
Materials and Methods
Humanized H2L5 Antibody
[0199] H2L5 is a humanized variant of the murine mAb clone 422.2
obtained from the lab of Daniel Olive, Institut Paoli-Calmettes,
INSERM (Marseille, France). The murine antibody was generated using
standard hybridoma technology by immunizing BALB/c mice
intraperitoneally with recombinant human ICOS-Fc using COS7
cells.
Cell Lines and Primary Cell Cultures
[0200] Murine tumor cell lines EMT6 (ATCC # CRL-2755) and CT26
(ATCC # CRL-2638) and human cell lines A549 (ATCC # CCL-185) A2058
(ATCC # CRL-11147), HCT116 (ATCC # CCL-247) were expanded and
frozen upon receipt and used at low passage (.ltoreq.10 passages)
for inoculation to mice. Prior to in vivo use, cell lines were
tested by PCR and confirmed negative for pathogens including
mycoplasma using the mouse/rat comprehensive CLEAR panel (Charles
River Research Animal Diagnostic Services).
[0201] All patient material was obtained with the appropriate
informed written consent in accordance with the GSK human
biological sample management (HBSM) policy and SOP. Whole blood in
sodium heparin tubes (BD Biosciences) and surgically resected tumor
tissues from cancer patients were obtained from Avaden Biosciences
(Seattle) shipped overnight by post. Primary T cells or PBMC from
healthy human donors were purified from whole blood collected in
sodium heparin tubes at the GSK on-site blood donation units with
appropriate consent and in accordance with the GSK HBSM policy.
PBMC were isolated by density gradient centrifugation through
Histopaque. T-cells were isolated by negative selection using
Dynabeads.TM. Untouched.TM. Human T-cell kit (Life Technologies) or
RosetteSep human CD4 or CD8 T-cell enrichment kits (StemCell) for
binding and functional assays. Isolated T cells were pre-activated
with plate-bound anti-CD3 (clone OKT3, eBioscience) and anti-CD28
(clone CD28.2, eBioscience) for 48-96 hrs to upregulate ICOS
expression.
Mice, Tumor Challenge and Treatment
[0202] All studies were conducted in accordance with the GSK Policy
on the Care, Welfare and Treatment of Laboratory Animals and were
reviewed by the Institutional Animal Care and Use Committee either
at GSK or by the ethical review process at the institution where
the work was performed. 6-8 week old female BALB/c mice
(Harlan/Envigo) were utilized for in vivo studies in a fully
accredited AAALAC facility. 5.times.10.sup.4 cells/mouse CT26 mouse
colon carcinoma or 1.times.10.sup.5 EMT6 mouse mammary carcinoma
tumor cells were inoculated subcutaneously into the right flank.
Prior to initiation of treatment, mice (n=10/treatment group) were
randomized with the Study Director software package (Studylog
Systems) when the tumors reached 100 mm.sup.3 unless otherwise
specified. ANOVA was used to ensure similarity between groups
(P>0.9).
[0203] The study investigator was blinded during the group
allocation and assessed the final outcome to ensure that group
distributions were acceptable for study initiation (P>0.9).
[0204] Based on inter-individual variability in tumor growth rates
from 5 separate studies in the CT26 syngeneic model, 10 mice per
group were justified as the optimal number necessary to observe an
effect size of approximately 0.8 between control and drug-treated
groups and to generate statistically significant data.
[0205] Tumor bearing mice received the mouse anti-ICOS (clone
7E.17G9) in different isotype backgrounds or H2L5 and/or mouse
anti-PD-1 (clone RMP1-14) or an isotype control in saline via
intraperitoneal injection twice weekly starting on randomization
day for a total of 6 doses. Tumor measurement of greater than 2,000
mm.sup.3 for an individual mouse and/or development of open
ulcerations resulted in mice being removed from study.
Binding Studies
[0206] The affinity and kinetics of H2L5 binding to rabbit
Fc-tagged recombinant extracellular human or cynomolgus ICOS
(generated in-house) was determined using a Biacore.TM. T200 (GE
Healthcare.TM.). The ICOS binding data was fitted to a 1:1 kinetics
model using the T200 data analysis software. Cell surface binding
of H2L5 to both freshly isolated unactivated and CD3/CD28 activated
CD4 and CD8 T cells was determined via detection of anti-human IgG,
kappa light chain FITC (Sigma) binding to H2L5 by flow
cytometry.
Antibodies
[0207] The following anti-human antibodies were used for flow
cytometry analysis, CD4 (RPA-T4, BD Biosciences), CD8 (RPA-T8,
Biolegend), CD69 (FN50, Biolegend), OX40 (ACT-35, eBioscience),
Ki67 (B56, BD Biosciences), ICOS (ISA3, eBioscience). The following
anti-mouse antibodies were used for flow cytometry analysis: CD3
(145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), CD8
(53-6.7, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7,
Biolegend), CD62L (MEL14, BD Biosciences), FOXP3 (Fjk-16s,
eBioscience), ICOS (C398.4a, Biolegend), Ki67 (16A8, Biolegend).
Apoptotis was measured using the Annexin V kit with 7-AAD
(Biolegend). For flow cytometry analysis of the human PBMC mouse
model the following antibodies were used: CD45 (HI30, BD
Biosciences), CD3 (UCHT1, Biolegend), CD4 (SK3, BD Biosciences),
CD45RO (UCHL1, Biolegend), CD62L (SK11, BD Biosciences). p-AKT
(S473, #4060 and T308, #13038), total Akt (#9272), pGSK3-.alpha.
(#5558), total GSK3-.alpha. (#12456), pS6 (S235/236, #2211 and
S240/244, #5364), total S6 (#2317), and pERK (#9101) (all from Cell
Signaling Technology) were used for Western Blots.
ADCC Assays
[0208] Whole PBMC or NK depleted PBMC were activated with
plate-bound anti-CD3 and anti-CD28 antibodies. Cells were incubated
with anti-ICOS antibodies (H2L5 IgG1, H2L5 IgG4PE and H2L5
Fc-disabled) or control antibodies at 10 .mu.g/mL final
concentration for 24 hours. Cells were stained with anti-CD8 and
CD4 antibodies followed by incubation with NIR Live/Dead dye
(Invitrogen). Stained cells were analyzed by flow cytometry
(FACSCanto, BD Biosciences) to measure T-cell killing based on NIR
Live/Dead cell dye staining.
[0209] In the Fc.gamma.RIIIa engagement reporter bioassay
(Promega), anti-CD3/CD28 pre-activated CD4 T cells were incubated
with the anti-ICOS and control antibodies for 45 minutes prior to
the addition of Jurkat-Fc.gamma.RIIIA-NFAT-luciferase effector
cells at an E:T cell ratio of 6:1. ONE-GLO luciferase reagent was
added to each well after 6 hrs of treatment and luminescence
intensity measured to determine engagement between the target T
cells and the effector cells on a Victor plate reader (Perkin
Elmer). CD4, CD8 and T.sub.reg populations were purified from
either donor PBMC pre-activated with anti-CD3/CD28 or disaggregated
tumor cells and tested directly ex vivo at 6:1 E:T ratio in
presence of IgG1 or IgG4PE H2L5 antibodies.
Functional Assays
[0210] H2L5 was tested in human PBMC assays either in a plate-bound
format with concurrent CD3 stimulation using freshly isolated PBMC
or in a soluble format in CD3/CD28 pre-stimulated PBMC as described
earlier. For PBMC from cancer patients, an overnight rest step was
included prior to treatment initiation. 10 .mu.g/mL soluble
pembrolizumab was used in in vitro assays to study effects of
combination. Cytokine concentrations in supernatants from these
assays were measured using bespoke human multiplex meso-scale
detection (MSD) kits (Meso Scale Diagnostics).
[0211] Human monocytes were isolated from whole blood of healthy
human donors, using CD14 MicroBeads (Miltenyi Biotec) for the T
cell:monocyte mixed culture assays. T cell and monocytes were donor
matched. CD3/CD28 pre-stimulated T cells and monocytes were mixed
at 2:1 ratio in AIM-V serum-free media and cultured together with
anti-CD3 dynabeads (Life Technologies), 100 IU of recombinant human
IL-2 and 100 ng/ml of M-CSF (Peprotech) prior to incubating with
soluble H2L5 or other control antibodies at 37.degree. C. for 4
days. 20 .mu.g/mL human Fc block (B564220) (BD biosciences) or
anti-CD32 mAb (MCA1075EL, Clone AT10) (AbD serotec) were used to
test the role of Fc.gamma.R cross linking.
[0212] For the MLR assays, monocytes (Lonza, Switzerland) were
grown in GM-CSF and IL-4 (Pepro Tech) supplemented LGM-3 media
(Lonza) for 9 days for differentiating into mDCs and TNF.alpha.
(R&D Systems) for an additional day before use in the MLR
assay. The mDC-T cell (1:10 ratio) mix was treated with 10 .mu.g/mL
soluble H2L5 Fc-disabled or the isotype control antibodies either
in the presence of anti-CD3 beads at a 1:10 bead to cell ratio
(Life Technologies) or CEFT peptide mix (0.02 .mu.g/mL) (JPT
Peptide Technologies) for 4 days before collecting the supernatants
for cytokine analysis by MSD.
[0213] Primary patient tumors were dissociated using GentleMACS
(Miltenyi Biotec) tissue dissociator. TIL were expanded in IL-2
supplemented RPMI media (Baldan et al., 2015) before treating with
anti-CD3 plus H2L5. Alternatively, tumor dissociated cells were
directly cultured ex vivo for up to 6 days following stimulation
with anti-CD3 plus H2L5 with 100 ng/ml IL-2 added after 24
hours.
[0214] For PBMC assays testing different H2L5 isotypes, anonymized
leukocyte cones from healthy donors were obtained from the National
Blood Service at Southampton General Hospital, UK and used within 4
hours. Use of human samples was approved by local ethical
committees in accordance with the Declaration of Helsinki. PBMC
were isolated by density gradient centrifugation (Lymphoprep) and
cultured in RPMI medium 1640 (Life Technologies) supplemented with
glutamine (2 mM), sodium pyruvate (1 mM), penicillin, and
streptomycin (100 IU/mL) at 37.degree. C. in 5% CO.sub.2.
[0215] Proliferation assays were performed as detailed previously
(35). Briefly, fresh PBMC were labelled with 1 .mu.M
carboxyfluorescein succinimidyl ester (CFSE) and cultured at high
density (1.times.10.sup.7/mL) for 48 hours prior to antibody
stimulations. For the PBMC stimulation, cells were transferred into
round-bottomed 96-well plates at 1.times.10.sup.5 per well and
stimulated with 1 .mu.g/ml OKT3 (plate-bound) and 5 .mu.g/ml
(soluble) H2L5 mAbs. On day 6, cells were labelled with
anti-CD8-e450 (SK-1, eBioscience) and anti-CD4-APC (RPA-T4, Insight
Biotechnology), and proliferation assessed by CFSE dilution on a
FACSCantoII flow cytometer (BD Biosciences). Results are expressed
as % divided cells compared to the unstimulated cells. NK depletion
was performed using CD56 micro beads (Miltenyi Biotec) according to
the manufacturer's instructions post 48 hours high density culture
(Hussain et al. Blood 2014).
Immunofluorescence Studies
[0216] Unstimulated and CD3/CD28 stimulated T cells were Fc blocked
with 20 .mu.g/mL human Fc block (B564220) (BD biosciences) or
anti-CD32 mAb (MCA1075EL, Clone AT10) (AbD serotec) to test the
role of Fc.gamma.R cross linking and then treated with 6 .mu.g/mL
cold labeled antibody (anti-ICOS or IgG4PE isotype control) on ice
for 1 hr. Cells were washed in cold buffer and transferred to
37.degree. C. for various times (0, 5, 15, 30 minutes and 1 hour)
to allow protein trafficking before fixing with freshly prepared 4%
paraformaldehyde (Sigma). Samples 1 or 2 hours after the initial
pulse at 37.degree. C. were re-pulsed with Alexa Fluor 647 labeled
anti-ICOS for 30 minutes at 37.degree. C., washed and fixed in
paraformaldehyde. The cells were transferred to Poly-L-lysine
coated coverslips for 15 minutes and then mounted on slides in
ProLong Gold with DAPI (Invitrogen). Analysis of the samples was
performed using a ZEISS LSM510 Meta Confocal microscope with a
63.times. oil immersion lens.
Human T-Cell Gene Expression
[0217] Whole blood was obtained from healthy volunteer donors (n=6)
at the GSK on-site Blood Donation Unit and T cells were purified
using RosetteSep.TM. Human T-Cell Enrichment Cocktail (Stemcell
Technologies) as described above. The cells were re-suspended
(5.times.10.sup.6 cells/mL) in AIM-V culture media (Gibco) and
incubated in 96-well plates (Falcon) that were sequentially
pre-coated with 0.6 .mu.g/mL of mouse anti-human CD3 mAb
(eBioscience) and 10 .mu.g/mL of anti-human ICOS or corresponding
isotype control mAbs--mouse IgG2 .alpha. .kappa. (eBioscience) and
IgG4PE. After 24 hours of incubation at 37.degree. C. and 5%
CO.sub.2, cells were pelleted, suspended in RLT buffer (Qiagen),
and stored at -80.degree. C. for RNA isolation. Total RNA was
extracted using the RNeasy Mini QIAcube Kit (Qiagen). RNA
expression levels were determined by NanoString nCounter Analysis
System. 50 ng of RNA was used in each reaction for gene signature
using NanoString Human PanCancer Immune profiling CodeSet according
to the manufacturer's instructions. Raw data was normalized using
built-in positive controls and house-keeping genes (nCounter
Expression Data Analysis Guide, NanoString). ArrayStudio (OmicSoft)
and GraphPad Prism (GraphPad Software) were used for further
analysis and graphs.
ICOS/ICOS-L Competition Assay
[0218] MSD plates were incubated overnight at 4.degree. C. with 10
.mu.g/mL recombinant ICOS protein (R&D Systems) diluted in PBS.
Plates were washed and blocked before adding isotype control or
H2L5 in a 7-point dose curve. After overnight incubation and
washes, the plates were incubated with 1 .mu.g/mL human ICOS ligand
(B7-H2) (R&D Systems) followed by incubation with 10 .mu.g/mL
biotinylated anti-human ICOS ligand (B7-H2) (R&D Systems)
antibody. Sulfo-tagged streptavidin at 10 .mu.g/mL in Diluent 100
was used for detection of the biotinylated ligand. The plates were
read immediately following MSD Read buffer addition on a MSD MESO
Quick Plex SQ 120 and data analyzed on MSD workbench software. Flow
cytometry was also used to investigate competition between cell
surface ICOS expressed by anti-CD3/CD28 activated T cells and
ICOS-L by H2L5. Activated T cells were incubated with different
concentrations of recombinant ICOS-L and then incubated with H2L5
and MFI of ICOS CD4+ and CD8+ ICOS cells determined.
Human PBMC Mouse Model
[0219] Adult immunodeficient NOD/SCID/IL-2R.gamma.null (NSG) mice
(Jackson Labs) were injected with human PBMC (20.times.10.sup.6 per
mouse) by intravenous injection via the tail vein. Mice were
implanted with human tumor cell lines A2058, A549, HCT116
(1.times.10.sup.6) 1-3 days post human PBMC injection; mice were
administrated isotype control or anti-human ICOS antibodies at
doses ranging from 0.004 mg/kg to 1.2 mg/kg by intraperitoneal
injection twice weekly for 3 weeks. Tumor bearing mice received the
mouse anti-ICOS (clone 7E. 17G9) in different isotype backgrounds
or H2L5 and/or Pembrolizumab (Merck; NDC #0006-3026-02) antibodies
or isotype controls in saline via intraperitoneal injection twice
weekly starting on randomization day for a total of 6 doses. Tumor
measurement of greater than 2,000 mm.sup.3 for an individual mouse
and/or development of open ulcerations resulted in mice being
removed from study.
[0220] Spleens and whole blood were collected post-euthanization at
24 hrs post 2.sup.nd or 4.sup.th dose of antibodies. Splenocytes
were isolated by mechanical dissociation followed by RBC lysis with
LCK lysis buffer (Lonza) and antibody staining whereas whole blood
was stained with the appropriate antibodies before RBC lysis with
FACSlyse (BD Biosciences). All samples were evaluated by flow
cytometry on FACScanto (BD) as described below.
Western Blotting
[0221] Activated T cells were treated with H2L5 or an isotype
control for up to 48 hours. CD4+ T cells were prestimulated with
CD3/CD28 Dynabeads.RTM. (ThermoFisher) at a cell-to-bead ratio of
1:20 for 48 hours, allowed to rest in the absence of stimulation
for 24 hours, and then treated with isotype control antibody or
H2L5 (10 .mu.g/mL) in the presence of plate-bound anti-CD3
antibody. Cells were lysed with cell lysis buffer (Cell Signaling
Technologies) containing protease and phosphatase inhibitors
(Roche). 25-30 .mu.g of protein was run on 4-12% Bis-Tris gels
(Invitrogen) and transferred onto nitrocellulose membranes
(Invitrogen). Membranes were blocked using LI-COR Odyssey Blocking
Buffer and subsequently immunoblotted using the primary and
secondary antibodies and scanned on a LI-COR Odyssey imaging
system.
FACS Analysis
[0222] Non-specific binding on activated T-cells was blocked by
incubation with human or mouse Fc block (Miltenyi Biotec) as
appropriate prior to the incubation with detection antibodies to
cell surface markers conjugated to different fluorophores on ice
for 30 minutes. For intracellular staining, the cells were fixed
and permeabilized using the Transcription Factor Buffer set (BD
biosciences). After compensation, data were acquired on FACS Canto
II or Fortessa (BD biosciences) and analyzed with FACSDiva (BD) or
Flowjo (Treestar) software.
Immunohistochemistry
[0223] Immunohistochemical detection of ICOS in non-small cell lung
cancer (NSCLC), breast cancer (BrCA) TNBrCa, and colorectal cancer
(CRC), was performed using a rabbit anti-human CD278 mAb (clone
SP98; Spring Biosciences) on a Leica Bond RX with associated
platform reagents. DAB (3, 3'-diaminobenzidine) was used for target
detection. Sections were counter stained with Hematoxylin (All
scale bars=20 .mu.m).
[0224] Clarient MultiOmyx platform (Neogenomics, California), a
multiplexed immunofluorescence (IF) assay was used to evaluate
expression of ICOS, PD-1, CD3, CD4 and CD8 among other T-cell
markers on FFPE tumor tissues obtained from vendors vetted by GSK
HBS group as described above. The iterative process included a
round of staining with a Cy3 and Cy5 conjugated antibody each and
imaging, followed by dye inactivation, background fluorescence
imaging and subtraction of the background before the repeating this
cycle for all markers in the panel.
Statistical Analysis
[0225] One-way ANOVA or Student's t-tests were used as specified in
the figure legend. Data were analyzed with GraphPad Prism software
(GraphPad) and p values of <0.05 were considered statistically
significant. (*P.ltoreq.0.05; **P.ltoreq.0.01; ***P.ltoreq.0.005;
****P.ltoreq.0.0001).
Sequence CWU 1
1
2115PRTMus musculus 1Asp Tyr Ala Met His1 5217PRTMus musculus 2Leu
Ile Ser Ile Tyr Ser Asp His Thr Asn Tyr Asn Gln Lys Phe Gln1 5 10
15Gly312PRTMus musculus 3Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr Phe
Asp Val1 5 10410PRTMus musculus 4Ser Ala Ser Ser Ser Val Ser Tyr
Met His1 5 1057PRTMus musculus 5Asp Thr Ser Lys Leu Ala Ser1
569PRTMus musculus 6Phe Gln Gly Ser Gly Tyr Pro Tyr Thr1
57121PRTArtificial SequenceH2L5 Vh 7Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ala Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Leu Ile Ser Ile
Tyr Ser Asp His Thr Asn Tyr Asn Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly
Arg Asn Asn Tyr Gly Asn Tyr Gly Trp Tyr Phe Asp Val Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser 115 1208106PRTArtificial
SequenceH2L5 Vl 8Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Ile
Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Leu Glu Pro Glu65 70 75 80Asp Phe Ala Val Tyr Tyr
Cys Phe Gln Gly Ser Gly Tyr Pro Tyr Thr 85 90 95Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 1059168PRTHomo sapiens 9Met Lys Ser Gly Leu
Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys1 5 10 15Val Leu Thr Gly
Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30Phe His Asn
Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45Gln Gln
Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50 55 60Leu
Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu65 70 75
80Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu
85 90 95Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu
Ser 100 105 110Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly
Gly Tyr Leu 115 120 125His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu
Lys Phe Trp Leu Pro 130 135 140Ile Gly Cys Ala Ala Phe Val Val Val
Cys Ile Leu Gly Cys Ile Leu145 150 155 160Ile Cys Trp Leu Thr Lys
Lys Met 16510199PRTHomo sapiens 10Met Lys Ser Gly Leu Trp Tyr Phe
Phe Leu Phe Cys Leu Arg Ile Lys1 5 10 15Val Leu Thr Gly Glu Ile Asn
Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30Phe His Asn Gly Gly Val
Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45Gln Gln Phe Lys Met
Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50 55 60Leu Thr Lys Thr
Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu65 70 75 80Lys Phe
Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95Tyr
Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser 100 105
110Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu
115 120 125His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp
Leu Pro 130 135 140Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu
Gly Cys Ile Leu145 150 155 160Ile Cys Trp Leu Thr Lys Lys Lys Tyr
Ser Ser Ser Val His Asp Pro 165 170 175Asn Gly Glu Tyr Met Phe Met
Arg Ala Val Asn Thr Ala Lys Lys Ser 180 185 190Arg Leu Thr Asp Val
Thr Leu 19511288PRTHomo sapiens 11Met Gln Ile Pro Gln Ala Pro Trp
Pro Val Val Trp Ala Val Leu Gln1 5 10 15Leu Gly Trp Arg Pro Gly Trp
Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30Asn Pro Pro Thr Phe Ser
Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45Asn Ala Thr Phe Thr
Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60Leu Asn Trp Tyr
Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala65 70 75 80Ala Phe
Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95Val
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105
110Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu
Arg Val 130 135 140Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro
Ser Pro Ser Pro145 150 155 160Arg Pro Ala Gly Gln Phe Gln Thr Leu
Val Val Gly Val Val Gly Gly 165 170 175Leu Leu Gly Ser Leu Val Leu
Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190Ser Arg Ala Ala Arg
Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195 200 205Leu Lys Glu
Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly 210 215 220Glu
Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro225 230
235 240Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser
Gly 245 250 255Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp
Gly Pro Arg 260 265 270Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His
Cys Ser Trp Pro Leu 275 280 28512290PRTHomo sapiens 12Met Arg Ile
Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu1 5 10 15Asn Ala
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30Gly
Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40
45Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser
Ser65 70 75 80Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser
Leu Gly Asn 85 90 95Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp
Ala Gly Val Tyr 100 105 110Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp
Tyr Lys Arg Ile Thr Val 115 120 125Lys Val Asn Ala Pro Tyr Asn Lys
Ile Asn Gln Arg Ile Leu Val Val 130 135 140Asp Pro Val Thr Ser Glu
His Glu Leu Thr Cys Gln Ala Glu Gly Tyr145 150 155 160Pro Lys Ala
Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175Gly
Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185
190Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr
195 200 205Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala
Glu Leu 210 215 220Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn
Glu Arg Thr His225 230 235 240Leu Val Ile Leu Gly Ala Ile Leu Leu
Cys Leu Gly Val Ala Leu Thr 245 250 255Phe Ile Phe Arg Leu Arg Lys
Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270Gly Ile Gln Asp Thr
Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285Glu Thr
29013273PRTHomo sapiens 13Met Ile Phe Leu Leu Leu Met Leu Ser Leu
Glu Leu Gln Leu His Gln1 5 10 15Ile Ala Ala Leu Phe Thr Val Thr Val
Pro Lys Glu Leu Tyr Ile Ile 20 25 30Glu His Gly Ser Asn Val Thr Leu
Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45His Val Asn Leu Gly Ala Ile
Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55 60Asp Thr Ser Pro His Arg
Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu65 70 75 80Pro Leu Gly Lys
Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp 85 90 95Glu Gly Gln
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr 100 105 110Lys
Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr 115 120
125His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln
130 135 140Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val
Ser Val145 150 155 160Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu
Gly Leu Tyr Gln Val 165 170 175Thr Ser Val Leu Arg Leu Lys Pro Pro
Pro Gly Arg Asn Phe Ser Cys 180 185 190Val Phe Trp Asn Thr His Val
Arg Glu Leu Thr Leu Ala Ser Ile Asp 195 200 205Leu Gln Ser Gln Met
Glu Pro Arg Thr His Pro Thr Trp Leu Leu His 210 215 220Ile Phe Ile
Pro Phe Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val225 230 235
240Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp
245 250 255Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn
Ser Ala 260 265 270Ile14116PRTArtificial Sequence37A10S713 Vh 14Glu
Val Gln Leu Val Glu Ser Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10
15Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Trp
20 25 30Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val
Ser 35 40 45Asn Ile Asp Glu Asp Gly Ser Ile Thr Glu Tyr Ser Pro Phe
Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Thr 85 90 95Arg Trp Gly Arg Phe Gly Phe Asp Ser Trp
Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11515111PRTArtificial Sequence37A10S713 Vl 15Asp Ile Val Met Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr
Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ser Gly 20 25 30Ser Phe Asn
Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu
Leu Ile Phe Tyr Ala Ser Thr Arg His Thr Gly Val Pro Asp 50 55 60Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His His His Tyr
85 90 95Asn Ala Pro Pro Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105 1101610PRTArtificial Sequence37A10S713 Vh CDR1 16Gly Phe
Thr Phe Ser Asp Tyr Trp Met Asp1 5 101717PRTArtificial
Sequence37A10S713 Vh CDR2 17Asn Ile Asp Glu Asp Gly Ser Ile Thr Glu
Tyr Ser Pro Phe Val Lys1 5 10 15Gly188PRTArtificial
Sequence37A10S713 Vh CDR3 18Trp Gly Arg Phe Gly Phe Asp Ser1
51915PRTArtificial Sequence37A10S713 Vl CDR1 19Lys Ser Ser Gln Ser
Leu Leu Ser Gly Ser Phe Asn Tyr Leu Thr1 5 10 15207PRTArtificial
Sequence37A10S713 Vl CDR2 20Tyr Ala Ser Thr Arg His Thr1
5219PRTArtificial Sequence37A10S713 Vl CDR3 21His His His Tyr Asn
Ala Pro Pro Thr1 5
* * * * *